Aryl and heteroaryl propene amides, derivatives thereof and therapeutic uses thereof

ABSTRACT

Compounds useful as antiproliferative agents, radioprotective agents and cytoprotective agents, including, for example, anticancer agents, are provided according to formula I: wherein ring A, ring B, ,X, R 1 , R 2 , R 3 , a, and b are as defined herein.

This application is a 371 of PCT/US03126954, filed 08/28/2003.

FIELD OF THE INVENTION

The invention relates to compositions and methods for the treatment ofproliferative disorders, including but not limited to cancer. Theinvention further relates to compositions that afford protection fromthe cytotoxic effects of ionizing radiation and of cytotoxicchemotherapeutic agents.

BACKGROUND OF THE INVENTION

Aryl and Heteroaryl Propene Amides

Cancer remains a leading cause of mortality in the United States and inthe world. To be useful, a new chemotherapeutic agent should have a widespectrum of activity and significant therapeutic index.

Aryl and heteroaryl propene amides have been prepared by reactingsubstituted aromatic acryloyl halides (such as cinnamoyl chlorides) withamino-substituted aromatic compounds (Kim et al., J. Chem. Soc. PerkinTrans. 2, 1995, p2257 describes reaction of cinnamoyl chlorides withanilines Alberghina et al., J. Org. Chem., Vol. 43, No. 6, 1978, p1122describes reaction of furylacryloyl and thienylacryloyl chlorides withanilines.). Another route to aryl and heteroaryl propene amides involvesreaction of an aromatic vinylhalide (preferably a bromide or iodide)with an aromatic amine using palladium catalysis (U.S. Pat. No.3,988,358).

Certain propene amides have shown activity in treatment ofatherosclerosis (Japanese patent 2001139550). Certain propene amideshave also shown biological activity as inhibitors of oviposition inseveral herbivorous insect species (Blaakmeer et al, Journal of NaturalProducts, Vol. 57, No. 1, pg. 90, 1994, and Vol. 57, No. 8, pg. 1145).Some substituted cinnamanilides have demonstrated bacteriostaticproperties (U.S. Pat. No. 3,975,435). Certain ortho-acyl -substitutedcinnamanilides have been shown to inhibit the proliferation of vascularintimal cells which occurs incident to vascular stenosis (EP 0 855 387A1). Other ortho acyl cinnamanilides have demonstrated biologicalactivity as antiallergy agents (U.S. Pat. No. 4,337,270). N-imidazolylcinnamides bearing an ortho-acyl substituent have been investigated asneovascularization inhibitors (Japanese patent applications 09363359 and10307760). Other related compounds, N-indolylcinnamides having an orthoacyl substituent, have been shown to possess activity as COX-2inhibitors (U.S. Pat. No. 6,300,363 B1). At least one cinnamanilide hasbeen shown to be an antagonist at the platelet-activating factor (PAF)receptor (Lamotte-Brasseur et al., Lipids, Vol. 26, No. 12, 1991,p1167). Also N-pyridyl cinnamides have demonstrated antagonist activityof the enzyme CADH (Duran et al., Bull. Soc. Chim. Fr., 1987, No. 4,1987, p 672).

Cell antiproliferative agents, and anticancer therapeutics inparticular, are needed which are useful in inhibiting proliferation ofand/or killing cancer cells. In particular, such agents are needed whichare selective in the killing of proliferating cells such as tumor cells,but not normal cells. Antineoplastic agents are needed which areeffective against a broad range of tumor types.

Ionizing Radiation Health Risks

Ionizing radiation has an adverse effect on cells and tissues, primarilythrough cytotoxic effects. In humans, exposure to ionizing radiationoccurs primarily through therapeutic techniques (such as anticancerradiotherapy) or through occupational and environmental exposure.

A major source of exposure to ionizing radiation is the administrationof therapeutic radiation in the treatment of cancer or otherproliferative disorders. Depending on the course of treatment prescribedby the treating physician, multiple doses may be received by a subjectover the course of several weeks to several months.

Therapeutic radiation is generally applied to a defined area of thesubject's body which contains abnormal proliferative tissue, in order tomaximize the dose absorbed by the abnormal tissue and minimize the doseabsorbed by the nearby normal tissue. However, it is difficult (if notimpossible) to selectively administer therapeutic ionizing radiation tothe abnormal tissue. Thus, normal tissue proximate to the abnormaltissue is also exposed to potentially damaging doses of ionizingradiation throughout the course of treatment.

There are also some treatments that require exposure of the subject'sentire body to the radiation, in a procedure called “total bodyirradiation”, or “TBI.” The efficacy of radiotherapeutic techniques indestroying abnormal proliferative cells is therefore balanced byassociated cytotoxic effects on nearby normal cells. Because of this,radiotherapy techniques have an inherently narrow therapeutic indexwhich results in the inadequate treatment of most tumors. Even the bestradiotherapeutic techniques may result in incomplete tumor reduction,tumor recurrence, increasing tumor burden, and induction of radiationresistant tumors.

Numerous methods have been designed to reduce normal tissue damage whilestill delivering effective therapeutic doses of ionizing radiation.These techniques include brachytherapy, fractionated andhyperfractionated dosing, complicated dose scheduling and deliverysystems, and high voltage therapy with a linear accelerator. However,such techniques only attempt to strike a balance between the therapeuticand undesirable effects of the radiation, and full efficacy has not beenachieved.

For example, one treatment for subjects with metastatic tumors involvesharvesting their hematopoietic stem cells and then treating the subjectwith high doses of ionizing radiation. This treatment is designed todestroy the subject's tumor cells, but has the side effect of alsodestroying their normal hematopoietic cells. Thus, a portion of thesubject's bone marrow (containing the hematopoietic stem cells), isremoved prior to radiation therapy. Once the subject has been treated,the autologous hematopoietic stem cells are returned to their body.

However, if tumor cells have metastasized away from the tumor's primarysite, there is a high probability that some tumor cells will contaminatethe harvested hematopoietic cell population. The harvested hematopoieticcell population may also contain neoplastic cells if the subject suffersfrom cancers of the bone marrow such as the variousFrench-American-British (FAB) subtypes of acute myelogenous leukemias(AML), chronic myeloid leukemia (CML), or acute lymphocytic leukemia(ALL). Thus, the metastasized tumor cells or resident neoplastic cellsmust be removed or killed prior to reintroducing the stem cells to thesubject. If any living tumorigenic or neoplastic cells are reintroducedinto the subject, they can lead to a relapse.

Prior art methods of removing tumorigenic or neoplastic cells fromharvested bone marrow are based on a whole-population tumor cellseparation or killing strategy, which typically does not kill or removeall of the contaminating malignant cells. Such methods includeleukopheresis of mobilized peripheral blood cells, immunoaffinity-basedselection or killing of tumor cells, or the use of cytotoxic orphotosensitizing agents to selectively kill tumor cells. In the bestcase, the malignant cell burden may still be at 1 to 10 tumor cells forevery 100,000 cells present in the initial harvest (Lazarus et al. J. ofHematotherapy, 2(4):457-66, 1993).

Thus, there is needed a purging method designed to selectively destroythe malignant cells present in the bone marrow, while preserving thenormal hematopoietic stem cells needed for hematopoietic reconstitutionin the transplantation subject.

Exposure to ionizing radiation can also occur in the occupationalsetting. Occupational doses of ionizing radiation may be received bypersons whose job involves exposure (or potential exposure) toradiation, for example in the nuclear power and nuclear weaponsindustries. Military personnel stationed on vessels powered by nuclearreactors, or soldiers required to operate in areas contaminated byradioactive fallout, risk similar exposure to ionizing radiation.Occupational exposure may also occur in rescue and emergency personnelcalled in to deal with catastrophic events involving a nuclear reactoror radioactive material. Other sources of occupational exposure may befrom machine parts, plastics, and solvents left over from themanufacture of radioactive medical products, smoke alarms, emergencysigns, and other consumer goods. Occupational exposure may also occur inpersons who serve on nuclear powered vessels, particularly those whotend the nuclear reactors, in military personnel operating in areascontaminated by nuclear weapons fallout, and in emergency personnel whodeal with nuclear accidents. Environmental exposure to ionizingradiation may also result from nuclear weapons detonations (eitherexperimental or during wartime), discharges of actinides from nuclearwaste storage and processing and reprocessing of nuclear fuel, and fromnaturally occurring radioactive materials such as radon gas or uranium.There is also increasing concern that the use of ordnance containingdepleted uranium results in low-level radioactive contamination ofcombat areas.

Radiation exposure from any source can be classified as acute (a singlelarge exposure) or chronic (a series of small low-level, or continuouslow-level exposures spread over time). Radiation sickness generallyresults from an acute exposure of a sufficient dose, and presents with acharacteristic set of symptoms that appear in an orderly fashion,including hair loss, weakness, vomiting, diarrhea, skin burns andbleeding from the gastrointestinal tract and mucous membranes. Geneticdefects, sterility and cancers particularly bone marrow cancer) oftendevelop over time. Chronic exposure is usually associated with delayedmedical problems such as cancer and premature aging. An acute a totalbody exposure of 125,000 millirem may cause radiation sickness.Localized doses such as are used in radiotherapy may not cause radiationsickness, but may result in the damage or death of exposed normal cells.

For example, an acute total body radiation dose of 100,000-125,000millirem (equivalent to 1 Gy) received in less than one week wouldresult in observable physiologic effects such as skin burns or rashes,mucosal and GI bleeding, nausea, diarrhea and/or excessive fatigue.Longer term cytotoxic and genetic effects such as hematopoietic andimmunocompetent cell destruction, hair loss (alopecia),gastrointestinal, and oral mucosal sloughing, venoocclusive disease ofthe liver and chronic vascular hyperplasia of cerebral vessels,cataracts, pneumonites, skin changes, and an increased incidence ofcancer may also manifest over time. Acute doses of less than 10,000millirem (equivalent to 0.1 Gy) typically will not result in immediatelyobservable biologic or physiologic effects, although long term cytotoxicor genetic effects may occur.

A sufficiently large acute dose of ionizing radiation, for example500,000 to over 1 million millirem (equivalent to 5-10 Gy), may kill asubject immediately. Doses in the hundreds of thousands of millirems maykill within 7 to 21 days from a condition called “acute radiationpoisoning.” Reportedly, some of the Chernobyl firefighters died of acuteradiation poisoning, having received acute doses in the range of200,000-600,000 millirem (equivalent to 2-6 Gy). Acute doses belowapproximately 200,000 millirem do not result in death, but the exposedsubject will likely suffer long-term cytotoxic or genetic effects asdiscussed above.

Acute occupational exposures usually occur in nuclear power plantworkers exposed to accidental releases of radiation, or in fire andrescue personnel who respond to catastrophic events involving nuclearreactors or other sources of radioactive material. Suggested limits foracute occupational exposures in emergency situations were developed bythe Brookhaven National Laboratories, and are given in Table 1.

TABLE 1 Whole Body Conditions for Dose Limit Activity RequiredConditions for Exposure  10,000 millirem* Protect property Voluntary,when lower dose not practical  25,000 millirem Lifesaving Operation;Voluntary, when lower Protect General Public dose not practical >25,000millirem Lifesaving operation; Voluntary, when lower Protect large dosenot practical, and population the risk has been clearly explained*100,000 millirem equals one sievert (Sv). For penetrating radiationsuch as gamma radiation, one Sv equals approximately one Gray (Gy).Thus, the dosage in Gy can be estimated as 1 Gy for every 100,000millirem.

A chronic dose is a low level (i.e., 100-5000 millirem) incremental orcontinuous radiation dose received over time. Examples of chronic dosesinclude a whole body dose of ˜5000 millirem per year, which is the dosetypically received by an adult working at a nuclear power plant. Bycontrast, the Atomic Energy Commission recommends that members of thegeneral public should not receive more than 100 millirem per year.Chronic doses may cause long-term cytotoxic and genetic effects, forexample manifesting as an increased risk of a radiation-induced cancerdeveloping later in life. Recommended limits for chronic exposure toionizing radiation are given in Table 2.

TABLE 2 Annual Occupational Organ or Subject Dose in millirem Whole Body  5000 Lens of the Eye 15,000 Hands and wrists 50,000 Any individualorgan 50,000 Pregnant worker 500/9 months Minor (16-18) receivingtraining   100

By way of comparison, Table 3 sets forth the radiation doses from commonsources.

TABLE 3 Sources Dose In Millirem Television <1/yr Gamma Rays, Jet CrossCountry 1 Mountain Vacation - 2 week 3 Atomic Test Fallout 5 U.S. Water,Food & Air (Average) 30/yr Wood 50/yr Concrete 50/yr Brick 75/yr ChestX-Ray 100 Cosmic Radiation (Sea Level) 40/yr (add 1 millirem/100 ftelev.) Natural Background San Francisco 120/yr Natural Background Denver50/yr Atomic Energy Commission Limit 5000/yr For Workers Complete DentalX-Ray 5000 Natural Background at Pocos de 7000/yr Caldras, Brazil WholeBody Diagnostic X-Ray 100,000 Cancer Therapy 500,000 (localized)Radiation Sickness-Nagasaki 125,000 (single doses) LD₅₀ Nagasaki &Hiroshima 400,000-500,000 (single dose)

Chronic doses of greater than 5000 millirem per year (0.05 Gy per year)may result in long-term cytotoxic or genetic effects similar to thosedescribed for persons receiving acute doses. Some adverse cytotoxic orgenetic effects may also occur at chronic doses of significantly lessthan 5000 millirem per year. For radiation protection purposes, it isassumed that any dose above zero can increase the risk ofradiation-induced cancer (i.e., that there is no threshold).Epidemiologic studies have found that the estimated lifetime risk ofdying from cancer is greater by about 0.04% per rem of radiation dose tothe whole body.

While anti-radiation suits or other protective gear may be effective atreducing radiation exposure, such gear is expensive, unwieldy, andgenerally not available to public. Moreover, radioprotective gear willnot protect normal tissue adjacent a tumor from stray radiation exposureduring radiotherapy. What is needed, therefore, is a practical way toprotect subjects who are scheduled to incur, or are at risk forincurring, exposure to ionizing radiation. In the context of therapeuticirradiation, it is desirable to enhance protection of normal cells whilecausing tumor cells to remain vulnerable to the detrimental effects ofthe radiation. Furthermore, it is desirable to provide systemicprotection from anticipated or inadvertent total body irradiation, suchas may occur with occupational or environmental exposures, or withcertain therapeutic techniques.

Pharmaceutical radioprotectants offer a cost-efficient, effective andeasily available alternative to radioprotective gear. However, previousattempts at radioprotection of normal cells with pharmaceuticalcompositions have not been entirely successful. For example, cytokinesdirected at mobilizing the peripheral blood progenitor cells confer amyeloprotective effect when given prior to radiation (Neta et al.,Semin. Radiat. Oncol. 6:306-320, 1996), but do not confer systemicprotection. Other chemical radioprotectors administered alone or incombination with biologic response modifiers have shown minor protectiveeffects in mice, but application of these compounds to large mammals wasless successful, and it was questioned whether chemical radioprotectionwas of any value (Naisin, J. R., Bacq and Alexander Award Lecture.“Chemical radioprotection: past, present, and future prospects”, Int JRadiat Biol. 73:443-50, 1998). Pharmaceutical radiation sensitizers,which are known to preferentially enhance the effects of radiation incancerous tissues, are clearly unsuited for the general systemicprotection of normal tissues from exposure to ionizing radiation.

What is needed are therapeutic agents to protect subjects who haveincurred, or are at risk for incurring exposure to ionizing radiation.In the context of therapeutic irradiation, it is desirable to enhanceprotection of normal cells while causing tumor cells to remainvulnerable to the detrimental effects of the radiation. Furthermore, itis desirable to provide systemic protection from anticipated orinadvertent total body irradiation, such as may occur with occupationalor environmental exposures, or with certain therapeutic techniques.

Protection from Toxic Side Effects of Experimental Chemotherapy

Experimental chemotherapy has been the mainstay of treatment offered topatients diagnosed with surgically unresectable advanced cancers, orcancers refractory to standard chemotherapy and radiation therapy. Ofthe more effective classes of drugs, curative properties are stilllimited. This is because of their relatively narrow therapeutic index,restricted dosage, delayed treatments and a relatively large proportionof only partial tumor reductions. This state is usually followed byrecurrence, increased tumor burden, and drug resistant tumors.

Several cytoprotective agents have been proposed to enhance thetherapeutic index of anticancer drugs. For methotrexate toxicity, suchagents include asparaginase, leucovorum factor, thymidine, andcarbipeptidase. Because of the extensive use of anthracyclines, specificand non-specific cytoprotective agents have been proposed which havevarying degrees of efficacy; included are corticosteroids, desrazoxaneand staurosporin. The latter is of interest in that it includes a G1/Srestriction blockade in normal cells. (Chen et al., Proc AACR 39:4436A,1998).

Cisplatin is widely used and has a small therapeutic index which hasspurred investigation and search of cytoprotectants. Among thecytoprotectants for cisplatin with clinical potential are mesna,glutathione, sodium thiosulfate, and amifostine (Griggs, Leuk. Res. 22Suppl 1:S27-33, 1998; List et al., Semin. Oncol. 23(4 Suppl 8):58-63,1996; Taylor et al., Eur. J Cancer 33(10):1693-8, 1997). None of theseor other proposed cytoprotectants such as oxonic acid forfluoropyrimidine toxicity, or prosaptide for paclitaxel PC12 celltoxicity, appears to function by a mechanism which renders normalreplicating cells into a quiescent state.

What is needed are effective cytoprotective agents which are effectivein protecting animals, inclusive of humans, from the cytotoxic sideeffects of chemotherapeutic agents.

The aryl and heteroaryl propene amide compounds of the present inventioninhibit tumor cell proliferation by inducing tumor cell death withoutkilling normal cells at therapeutically useful concentrations. Thecompounds of the present invention are effective against a broad rangeof tumor types. Without wishing to be bound by any theory, it isbelieved that the compounds affect the Mitogen Activated Protein Kinase(MAPK) signal transduction pathway, thereby affecting tumor cell growthand viability.

SUMMARY OF THE INVENTION

It is an object of the invention to provide compounds, pharmaceuticalcompositions and therapeutic methods. The biologically active compoundsare in the form of aryl and heteroaryl propene amides, and saltsthereof.

It is an object of the invention to provide compounds, compositions andmethods for the treatment and/or prevention of cancer and otherproliferative disorders.

It is an object of the invention to provide compounds which areselective in killing tumor cells but not normal cells at therapeuticallyuseful concentrations.

It is an object of the invention to provide compounds, compositions andmethods for inducing neoplastic cells to selectively undergo apoptosis.

It is a further object of this invention to provide compounds,compositions and methods which enable prophylactic treatment ofproliferative disorders.

It is a further object of this invention to provide compoundscompositions and methods for protecting normal cells and tissues fromthe cytotoxic and genetic effects of exposure to ionizing radiation, insubjects who have incurred, will in the future incur, or are at risk forincurring exposure to ionizing radiation.

The exposure to ionizing radiation may occur in controlled doses duringthe treatment of cancer and other proliferative disorders, or may occurin uncontrolled doses beyond the norm accepted for the population atlarge during high risk activities or environmental exposures.

It is an object of the invention to provide compositions and methods forprotecting individuals from the cytotoxic side effects ofchemotherapeutic agents, particularly mitotic phase cell cycleinhibitors and topoisomerase inhibitors, used in the treatment of cancerand other proliferative disorders.

It is an object of the invention provide a method for treating cancer orother proliferative disorder which reduces or eliminates cytotoxiceffects on normal cells.

It is an object of the invention to enhance the effects ofchemotherapeutic agents, particularly mitotic phase cell cycleinhibitors and topoisomerase inhibitors, used for the treatment ofcancer or other proliferative disorders.

It is an object of the present invention to provide a therapeuticprogram for treating cancer or other proliferative disorder whichincludes administration of a cytoprotective compound prior toadministration of a chemotherapeutic agent, which cytoprotectivecompound induces a reversible cycling quiescent state in non-tumoredtissues.

It is an object of the invention to provide a method for safelyincreasing the dosage of chemotherapeutic agents, particularly mitoticphase cell cycle inhibitors and topoisomerase inhibitors, used in thetreatment of cancer and other proliferative disorders.

In one aspect, the invention is directed to novel compounds of formulaI:

wherein:

ring A and ring B are independently selected from the group consistingof aryl and heteroaryl, provided that ring A is other than pyridyl,quinazolyl or naphthyridyl;

X is O or S, preferably O;

R¹ is independently selected from the group consisting of —R⁴;—SO₂(C₁-C₆)alkyl; C(═O)R⁴; —C(═O)OR⁴; —C(═O)O(C₁-C₆)alkylenearyl,preferably —C(═O)O(CH₂)aryl; —OR⁴; —(C₂-C₆)alkynyl;—(C₃-C₆)heteroalkenyl; —(C₂-C₆)alkylene-OR⁴; substituted aryl;unsubstituted aryl; substituted heteroaryl; unsubstituted heteroaryl;substituted aryl(C₁-C₃)alkyl; unsubstituted aryl(C₁-C₃) alkyl;substituted heteroaryl(C₁-C₃)alkyl and unsubstitutedheteroaryl(C₁-C₃)alkyl;

each R² is independently selected from —OR⁴; halogen, preferablyfluorine; —C≡N; —CO₂R⁴; —C(═O)NR⁴ ₂; —C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴; —(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂; —NHC(═O)(C₁-C₆)alkyl;sulfamyl; carbarnyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;—S(C₁-C₃)alkyl; —S(═O)(C₁-C₃)alkyl; and —SO₂(C₁-C₃)alkyl;

b is 1, 2, 3, 4or5;

provided that when B is phenyl, R² is other than 2,3-di-OR⁴, and3,4-di-OR⁴; and

provided that when R³ is halogen, R² is not chlorine, bromine or iodine;

indicates a single bond, whereby the compounds of formula I may be ineither the E or the Z conformation;

each R³ is independently selected from halogen; (C₁-C₆)alkyl; —OR⁴;—C≡N; —C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴; —(C₁-C₆)—OR⁴; nitro;phosphonato; —NHC(═O)(C₁-C₆)alkyl; sulfamyl; —OC(═O)(C₁-C₃)alkyl,preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably—O(C₂-C₆)—N (CH₃)₂; and (i) or (ii) below:

wherein:

each M is a bivalent connecting group independently selected from thegroup consisting of —(C₁-C₆)alkylene-, —(CH₂)_(d)- V—(CH₂)_(e)-,—(CH₂)_(f)- W—(CH₂)_(g)- and -Z-;

each y is independently selected from the group consisting of 0 and 1;

each V is independently selected from the group consisting of arylene,heteroarylene, —C(═O)—, —C(═O)(C₁-C₆)perfluoroalkylene, —C(═O)—,—C(═S)—, —S(═O)—, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴— and —SO₂NR⁴—;

each W is independently selected from the group consisting of —NR⁴—, —O—and —S—;

each d is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each e is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each f is independently selected from the group consisting of 1, 2 and3, preferably from 1 and 2;

each g is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

wherein the absolute stereochemistry of -Z- is S or R, or a mixture of Sand R;

each R^(α) is independently selected from the group consisting of —H,—(C₁-C₆) alkyl, —(CH₂)₃—NH—C(NH₂)(═NH), —CH₂C(═O)NH_(2,)—CH₂COOH,—CH₂SH, —(CH₂)₂C(═O)—NH₂, —(CH₂)₂COOH, —CH₂-(2-imidazolyl), —(CH₂)₄—NH₂,—(CH₂)₂—S—CH₃, phenyl, CH₂-phenyl, —CH₂—OH, —CH(OH)—CH₃,—CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl); and includes compounds whereinR^(α) and R⁴ combine to form a 5-, 6- or 7-membered heterocyclic orcarbocyclic ring;

a is 1, 2 or 3;

provided that when A is phenyl, R³ is other than 3,4,5-tri-OR⁴, and whenR² is 4-methoxy, R³ is other than 4-methoxy;

R⁴ is independently selected from the group consisting of —H, —(C₁-C₆)alkyl, substituted —(C₁-C₆)alkyl, —(C₂-C₆)alkenyl, substituted—(C₂-C₆)alkenyl and heteroalkyl, wherein two R⁴ groups may together forma heterocycle;

each R⁵ is independently selected from the group consisting of —R⁴;unsubstituted aryl; substituted aryl; substituted heterocyclic;unsubstituted heterocyclic; —CO₂R⁴; —C(═O)NR⁴ ₂; —C(═NH)—NR⁴ ₂;—(C₁-C₆)perfluoroalkyl; —CF₂Cl; —P(═O)(OR⁴)₂; —OP(═O)(OR⁴)₂; —CR⁴R⁷R⁸and a monovalent peptidyl moiety with a molecular weight of less than1000, preferably with a molecular weight of less than 800, morepreferably with a molecular weight of less than 600, most preferablywith a molecular weight of less than 400; provided that when y is 0 andR⁵ is —CO₂R⁴, then R⁴ is not —H;

each R⁶ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, and aryl(C₁-C₃)alkyl,

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, —C(═O)R⁸, —OR⁴, —SR⁴, —OC(═O)(CH₂)₂CO₂R⁶, guanidino, NR⁴₂, —NR⁴ ₃ ⁺, —N⁺(CH₂CH₂OR⁵)₃, halogen, phenyl, substituted phenyl,heterocyclyl, and substituted heterocyclyl; and

each R⁸ is independently selected from the group consisting of R^(α),halogen, —NR⁴ ₂ and heterocycles containing two nitrogen atoms;

-   -   wherein the substituents for the substituted aryl and        substituted heterocyclic groups comprising or included within        Ar, R¹, R⁵ R⁶, R⁷ and R^(α) are independently selected from the        group consisting of halogen; (C₁-C₆) alkyl; (C₁-C₆)alkoxy; —NO₂;        —C≡N; —C(═O)O(C₁-C₃)alkyl; —OR⁴; —(C₂-C₆) -OR⁴; phosphonato;        —NR₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl; carbamyl;        —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)        alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂; and        (C₁-C₃)perfluoroalkyl; or a salt of such a compound.

When R⁵ is a monovalent peptidyl moiety, the attachment point on thepeptidyl moiety may be via a carboxyl group or through an amino group.Further, the carboxyl or amino groups may be either terminalcarboxyl/amino groups or may be side chain groups such as, for example,the side chain amino group of lysine or the side chain carboxyl group ofaspartic acid. The attachment point on the peptidyl moiety willcorrelate with the particular selection of the M tether. Thus, for R⁵ asa peptidyl moiety of molecular weight less than 1000, it is providedthat:

(1) when V is —C(═O)—, —C(═S)—, —S(═O)—or —SO₂—, and e is 0, then thepeptidyl moiety is coupled to M through the peptide's amino terminus orthrough a sidechain amino group to form an amide, thioamide, sulfinamideor sulfonamide, respectively;

(2) when V is —C(═O)NR—, —SO₂NR⁴—, or —NR⁴—, and e is 0, then thepeptidyl moiety is coupled to M through the peptide's carboxy terminusor through a sidechain carboxyl group to form an imide, sulfonimide, orcarboxamide, respectively; and

(3) when W is —S— or —O—, and g is 0, then the peptidyl moiety iscoupled to M through the peptide's carboxy terminus or through asidechain carboxyl group to form a carbothioic acid ester or acarboxylic ester, respectively.

According to one sub-embodiment of the compounds of the invention, thereis provided a compound, wherein -Z- is:

wherein the absolute stereochemistry of -Z- is either S or R; and

each R^(α) is independently selected from the group consisting of —H,—CH₃, —(CH₂)₃—N—C(NH₂)(—NH), —CH₂C(═O)NH₂, —CH₂COOH, —CH₂SH,—(CH₂)2C(═O)—NH₂, —(CH₂)₂COOH, —CH₂-(2-imidazolyl), —CH(CH₃)—CH₂CH₃,—CH₂CH(CH₃)₂, —(CH₂)₄—NH₂, —(CH₂)₂—S—CH₃, phenyl, CH₂-phenyl, —CH₂—OH,—CH(OH)—CH₃, —CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl), —CH(CH₃)₂ and—CH₂CH₃; and includes compounds wherein R^(α) and R⁴ combine to form a5-, 6- or 7-membered heterocyclic ring.

Such compounds include, for example,

(E)-N-(4-methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;

(E)-N-(4-methoxy-3-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;

(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,3,4,5,6-pentafluorophenyl)-2-propenamide;

(E)-N-(4-bromophenyl)-3-(3-methoxy-4-fluorophenyl)-2-propenamide;

(E)-N-(4-bromophenyl)-3-(3-cyano-4-fluorophenyl)-2-propenamide;

(E)-N-(4-bromophenyl)-3-(3-carboxy-4-fluorophenyl)-2-propenamide;

(E)-N-(4-methoxy-3-nitrophenyl)-3-(3-fluoro-4-nitrophenyl)-2-propenamide;

(E)-N-(4-bromophenyl)-3-(2,4-difluorophenyl)-2-propenamide;

(E)-N-(4-methoxy-3-aminophenyl)-3-(3-fluoro-4-aminophenyl)-2-propenamide;and salts thereof.

According to a preferred sub-embodiment, there is provided a compound offormula I, wherein each V is independently selected from the groupconsisting of —C(═O)—, —C(═S)—, —S(═O)—, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴—and —SO₂NR⁴—.

According to a more preferred sub-embodiment, there is provided acompound of formula I, wherein

R² is independently selected from —OR⁴; —C≡N; —CO₂R⁴; —C(═O)NR⁴ ₂;—C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴; —(C₂-C₆)—OR⁴; phosphonato; —NR⁴₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl; carbamyl; —OC(═O)(C₁-C₃)alkyl,preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably—O(C₂-C₆)—N(CH₃)₂; —S(C₁-C₃)alkyl; —S(═O)(C₁-C₃)alkyl; and—SO₂(C₁-C₃)alkyl;

provided that when B is phenyl, R² is other than 2,3-di-OR⁴, 3,4-di-OR⁴,3,4,5-tri-OR⁴;

b is 1, 2, or 3; and

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl and —(C₁-C₆)acyl.

Such compounds include, for example,(E)-N-(4-methoxy-3-trifluoroacetamidophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide; and salts thereof.

According to a further preferred sub-embodiment, there is provided acompound of formula I, wherein each R⁵ is independently selected fromthe group consisting of —R⁴; unsubstituted aryl; substituted aryl;substituted heterocyclic; unsubstituted heterocyclic; —CO₂R⁴; —C(═O)NR⁴₂; —C(═NH)—NR⁴ ₂; and a monovalent peptidyl moiety with a molecularweight of less than 1000, preferably with a molecular weight of lessthan 800, more preferably with a molecular weight of less than 600, mostpreferably with a molecular weight of less than 400; provided that wheny is 0 and R⁵ is —CO₂R⁴, then R⁴ is not —H; and

-   -   wherein the substituents for the substituted aryl and        substituted heterocyclic groups comprising or included within        Ar, R¹, R⁵ and R^(α) are independently selected from the group        consisting of halogen; (C₁-C₆)alkyl; (C₁-C₆)alkoxy; —NO₂; —C≡N;        —C(═O)O(C₁-C₃)alkyl; —OR⁴; —(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂;        —NHC(═O)(C₁-C₆)alkyl; sulfamyl; carbamyl; —OC(═O)(C₁-C₃)alkyl,        preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably        —O(C₂-C₆)—N(CH₃)₂; and (C₁-C₃)perfluoroalkyl; or a salt of such        a compound.

According to another sub-embodiment of the compounds of the invention,there is provided a compound, wherein one R³ substituent, designatedR^(3p), is positioned in a substitution orientation relative to the

moiety which is closest to the planar angle formed by a para substituentin a six-membered aromatic ring and forms a planar angle with the

moiety of between about 135° and about 180°; or a salt of such acompound.

In such embodiments:

R^(3p) is selected from the group consisting of halogen; (C₁-C₆)alkyl;(C₁-C₆)alkoxy; —C≡N; —C(═O)NR⁴ ₂; —C(═NR⁴)NR⁴ ₂; —(C₁-C₃)alkylene-CO₂R⁴;—OR⁴; —(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;—OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂,preferably —O(C₂-C₆)—N(CH₃)₂; and (C₁-C₃)perfluoroalkyl.

Ring A, Ring B, X, M, d, e, f, g, V, W, Z, R¹, R², R⁴, R⁵, R⁶, R⁷, a, b,y, R^(α), and any remaining R³ substituents are as defined above forformula I.

Such compounds include, for example:

(E)-N-(4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;

(E)-N-(4-methoxyphenyl)-3-(2,6-dimethoxyphenyl)-2-propenamide;

(E)-N-(3-hydoxy-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;and salts thereof

According to another sub-embodiment, a compound of formula I is providedwherein at least one R³ substituent, designated R^(3m) is positioned ina substitution orientation relative to that of the

moiety which is closest to the dihedral angle formed by a metasubstituent in a six-membered aromatic ring and forms a planar anglewith the

moiety of between about 90° and about 145°.

Each R^(3m) is selected from the group consisting of nitro and (i) and(ii) below:

Ring A, Ring B, X, M, d, e, f, g, V, W, Z, R¹, R², R⁴, R⁵, R⁶, R⁷, a, b,y, R^(α), and any remaining R³ substituents are as defined for formula Iabove.

In one sub-embodiment of compounds of the invention, a compound offormula I is provided wherein ring A is phenyl, and wherein ring B, andM, d, e, f, g, V, W, Z, R¹, R², R³, R^(3m), R^(3p), R⁴, R⁵, R⁶, R⁷, a,b, y and R^(α) are as defined, as for formula I above; or a salt of sucha compound.

In another sub-embodiment of compounds of the invention, a compound offormula I is provided wherein ring B is phenyl, and wherein ring A, andM, d, f, g, V, W, Z, R¹, R², R³, R^(3m), R^(3p), R⁴ R⁵, R⁶, R⁷, a, b, yand R^(α) are as defined as for formula I above; or a salt of such acompound.

In a further sub-embodiment thereof, a compound of formula I is providedwherein ring A and ring B are phenyl, or a salt of such a compound.

In another embodiment of the invention, a compound of formula I, andsalts thereof, is provided wherein at least one R² substituentdesignated R^(2o), is located at a position on ring B, ortho to the

moiety; and wherein X, M, d, e, f, g, V, W, Z, R¹, R², R³, R^(3m),R^(3p), R⁴, R⁵, R⁶, R⁷, a, b, y and R^(α) are as defined as for formulaI above.

In another embodiment of the invention, there is provided a compoundaccording to the formula Ia:

wherein q is 0 or 1;

n is independently selected from 0 and 1; wherein the sum of n isselected from 1, 2 and 3; and

X, R¹, R², R^(2o), R^(3m) and R^(3p) are as defined as for formula Iabove;

or a salt thereof,

In a sub-embodiment thereof, there are provided a compound wherein eachR^(3m) is independently selected from the group consisting of (i) and(ii) below:

R^(3p) is selected from the group consisting of halogen; (C₁-C₆)alkyl;(C₁-C₆)alkoxy; —C≡N; —C(═O)NR⁴ ₂; —C(═NR⁴)NR⁴ ₂;—O(C₁-C₃)alkylene-CO₂R⁴; —OR⁴; —(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂;—NHC(═O)(C₁-C₆)alkyl; sulfamyl; —OC(═O)(C₁-C₃)alkyl, preferably—OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;and (C₁-C₃)perfluoroalkyl;

each R^(2o) is independently selected from the group consisting of(C₁-C₆)alkoxy; —NR⁴ ₂; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃; and—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;

R² is selected from the group consisting of halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, —NR⁴ ₂, —C≡N, —CO₂R⁴, —C(═O)NR⁴ ₂, —C(═NR⁴)NR⁴ ₂, andperfluoro(C₁-C₃)alkyl; and X, M, d, e, f, g, V, W, Z, R¹, R⁴, R⁵, R⁶,R⁷, R⁸, a, b, y, n, q and R^(α) are as defined above for formula I;

or a salt of such a compound.

Such compounds include, for example,(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide,and salts thereof

In a further sub-embodiment of compounds of the invention, a compound ofthe formula Ie, below, and salts thereof is provided:

wherein

R², R^(2o), R³m, R^(3p), q and n are as defined above for formula I.

Such compounds include:

(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide,and salts thereof.

In another embodiment of compounds of the present invention, there isprovided a compound of formula I

wherein:

X is S, and

Ring A, Ring B, R¹, R², R³ a and b substituents are as defined above forformula I;

or a salt of such a compound.

In a sub-embodiment thereof, there is provided a compound of formula Is

wherein R², R^(2o), n, R^(3p), R^(3m), M, y, and R⁵ are as defined abovefor formula Ic;

or a salt of such a compound.

According to other embodiments of the invention, processes for preparingcompounds according to formula I are provided.

In one such embodiment, a process for preparing a compound of formula I,or a salt thereof, wherein the olefin double bond is in the Econformation, is provided comprising:

(1) coupling a compound of formula II:

wherein A, R¹, R³, and a are defined as for formula I above;

with an alkyl ester of a malonic acid halide:

to yield a carboxylic ester compound of formula III:

(2) hydrolyzing the carboxylic ester compound of formula III to form acarboxylic acid compound of formula IV:

(3) coupling of the caboxylic acid compound of formula IV with anaromatic aldehyde of formula V:

wherein R², B and b are defined as in formula I above; in an acidicsolvent or acidic solvent mixture, particularly glacial acetic acid atelevated temperature to form a compound of formula I or a salt of such acompound.

In the aforesaid process, certain malonic acid halides, particularlymalonic acid chlorides, used in the initial acylation step, areavailable commercially and are also readily prepared by methods known toone of skill in the art. These methods include reaction of the precursormalonic acid mono ester with a chlorinating agent such as thionylchloride, phosphorous pentachloride or phosphorous trichloride. Otheracid halides are also synthetically accessible, including acid fluoridesvia reaction with, for example, hexafluoroacetone and acid bromides via;reaction with, for example, thionyl bromide. The alkyl ester moiety ofthe alkyl malonic acid halide is preferably a (C₁-C₁₀)alkyl ester, morepreferably a (C₁-C₆)alkyl ester, most preferably a commerciallyavailable reagent such as, for example, a methyl or ethyl ester.

The depiction of the malonic acid halide reagent shows a chlorineresidue as the leaving group. In addition to acid chlorides as shown,other leaving groups are possible and useful in this method includingfor example, fluorides, bromides and mixed anhydrides. The acid chlorideis preferred.

The hydrolysis of step 2 is performed in an aqueous base such as, forexample lithium hydroxide, sodium hydroxide or potassium hydroxide. Thesolvent medium may be aqueous or a mixture of water and a water-miscibleorganic solvent such as ethanol or tetrahydrofuran (THF).

According to a further embodiment of the invention, a process forpreparing a compound of formula I, or a salt thereof, is providedcomprising:

(1) halogenating a carboxylic acid of formula VI with a halogenatingagent:

to form an acid halide of formula VII:

(2) coupling the acid halide VII to an aromatic amino compound offormula II

to form an amide compound of formula I or a salt of such a compound.

The acid halide VII may be an acid fluoride, an acid chloride or an acidbromide. In addition, other activated carboxylic acids are useful inthis method, including for example mixed anhydrides and/or the use of acatalyzing reagent such as 4-dimethyl amino pyridine (DMAP).

The carboxylic acid (and the intermediate acid halide of formula VII) isshown in formula VI as being either in the E or the Z conformation, thusthis process is capable of producing compounds of formula I in which theolefin double bond is in either the E or the Z conformation.

In the step 2 coupling of the acid halide VII with the aromatic amineII, an acid scavenger such as, for example, triethyl amine (TEA) ordiisopropylethyl amine (DIPEA) is generally used to react with thebyproduct acid formed in the reaction, i.e., hydrochloric acid (HCl) inthe instance of reaction with an acid chloride.

For the synthesis of an acid halide such as VII, suitable halogenatingagents include thionyl chloride, thionyl bromide, phosphorouspentachloride, phosphorous oxychloride and hexafluoroacetone.

According to another embodiment of the invention, a process forpreparing a compound of formula I, or a salt thereof, is provided,comprising:

reacting an aromatic amino compound of formula II

with a carboxylic acid compound of formula VI:

and an amide coupling agent, to form a compound of formula I or a saltof such a compound.

In the aforesaid process of coupling compounds of formula II tocompounds of formula VI, “amide coupling reagents” are compounds used tocouple unactivated carboxylic acid moieties to amino groups, such as thearomatic amino moiety of a compound of formula I wherein —R^(3m) is NH₂(i.e., wherein —R^(3m) is formula (i), y is 0, R⁴ is —H and R⁵ isR⁴═—H). Such amide coupling reagents include for example, reagents suchas, for example, diisopropyl carbodiimide (DIC), dicyclohexylcarbodiimide (DCC) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate (HATU).

Also, the aromatic propionic acid is shown in formula VI as being eitherin the E- or the Z-conformation. This process is therefore capable ofproducing compounds of formula I in which the olefin double bond is ineither the E or the Z conformation.

Additionally, in the aforesaid process of coupling compounds of formulaII to compounds of formula VI, certain functional groups which would besensitive to the reaction conditions may be protected by protectinggroups. The term “protecting group” refers to a derivative of a chemicalfunctional group which is employed to derivatize chemicalfunctionalities which would otherwise be incompatible with theconditions of a desired reaction. The protecting group renders such afunctional group stable to the desired reaction conditions and may laterbe removed to regenerate the de-protected functionality. One example ofthe use of protecting groups is in the common reaction of the aminogroup of a first amino acid with the carboxyl group of a second aminoacid to form an amide bond. However, since each reactant contains bothan amino and a carboxylate functional group, the reaction between themis (1) nonspecific as to which amino group will react with whichcarboxyl group, and (2) subject to polymerization since the product ofthe reaction still contains both reactive moieties. A protecting groupon the carboxylate of the first amino acid and a protecting group acidon the amino group of the second amino acid will serve to limit thereagents to the single desired reaction of the amino group of the firstamino acid with the carboxylic acid of the second amino acid and yieldsa product which will not react further because both of the remainingreactive moieties are blocked by protecting groups which may besubsequently be selectively removed. The present reaction would reflectsuch a need for a protecting group if substitution on the A- or theB-ring included, for example, an amino group, or a carboxyl group inaddition to those desired to effect coupling to form the targetedpropene amide backbone.

Any chemical functionality that is a structural component of any of thereagents used to synthesize compounds of this invention may beoptionally protected with a chemical protecting group if such aprotecting group is useful in the synthesis of compounds of thisinvention. Appropriate protecting groups for amine functionalitiesinclude, for example, such moieties as tert-butoxy carbonyl (t-Boc),benzyl, 9-fluorenyl-methoxycarbonyl (Fmoc) or benzyloxycarbonyl (CBZ).Appropriate protecting groups for carboxyl groups include, for exampletert-butyl esters. Techniques for selecting, incorporating and removingchemical protecting groups may be found in “Protecting Groups In OrganicSynthesis” by Theodora Green, the entire disclosure of which isincorporated herein by reference.

In addition to use of a protecting group, sensitive functional groupsmay be introduced as synthetic precursors to the functional groupdesired in the final product. An example of this is an aromatic nitro(—NO₂) group. The aromatic nitro group goes not undergo any of thenucleophilic reactions of an aromatic amino group. However, the nitrogroup is essentially a protected amino group because it is readilyreduced to the amino group under mild conditions that are selective forthe nitro group over most other functional groups.

According to another embodiment of the invention, pharmaceuticalcompositions are provided, comprising a pharmaceutically acceptablecarrier and a compound according to formula I:

wherein:

ring A ring B, R¹, R², R³, X, a and b are as described above for FormulaI; or a salt of such a compound.

In another embodiment of compounds of the invention, there is provided aconjugate of the formula, I-L-Ab or Ic-L-Ab;

wherein,

I and Ic are compounds of formula I and formula Ic as defined herein;

Ab is an antibody; and

-L- is a single covalent bond or a linking group covalently linking saidcompound to said antibody.

In yet another embodiment of the invention, a conjugate of the formulaI-L-Ab is provided wherein I is a compound of formula I; Ab is anantibody; and -L- is a single bond or a linking group covalently linkingsaid compound of formula I to said antibody.

In a sub-embodiment thereof, a conjugate of the formula Ic-L-Ab isprovided wherein Ic is a compound of formula Ic; Ab is an antibody; and-L- is a single bond or a linking group covalently linking said compoundof formula Ic to said antibody.

In a preferred sub-embodiment of the aforesaid conjugates of theformulae I-L-Ab and Ic-L-Ab, said antibody (Ab) is a monoclonal antibodyor a monospecific polyclonal antibody.

In a more preferred sub-embodiment of the aforesaid conjugates of theformulae I-L-Ab and Ic-L-Ab, the aforesaid antibody (Ab) is atumor-specific antibody.

In yet a further embodiment of the present invention, there is provideda compound of formula I derivatized as a substrate for a β-lactanaseenzyme.

A pharmaceutical composition is additionally provided as describedsupra, comprising a pharmaceutically acceptable carrier and at least oneconjugate according to formulae I-L-Ab or fc-L-Ab.

According to another embodiment of the invention, there is provided amethod of treating an individual for a proliferative disorder,particularly cancer, comprising administering to the individual aneffective amount of at least one compound of formula I^(i):

wherein:

ring A and ring B are independently selected from the group consistingof aryl and heteroaryl;

X is O or S, preferably O;

R¹ is independently selected from the group consisting of —R⁴,—SO₂(C₁-C₆)alkyl, C(═O)R⁴, —C(═O)OR⁴, —C(═O)O(C₁-C₆)alkylenearyl, —OR⁴,—(C₂-C₆)alkynyl, —(C₃-C₆)heteroalkenyl, —(C₂-C₆)alkylene-OR⁴,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aryl(C₁-C₃)alkyl, unsubstitutedaryl(C₁-C₃)alkyl, substituted heteroaryl(C₁-C₃)alkyl and unsubstitutedheteroaryl(C₁-C₃)alkyl;

R² is independently selected from (C₁-C₆)alkyl; halogen; —OR⁴; —C≡N;—NO₂; —CO₂R⁴; —C(═O)NR⁴ ₂; C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴;—(C₂-C₆)—OR⁴; phosphonato; —NR₄ ²; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;carbamyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;—S(C₁-C₃)alkyl; —S(═O)(C₁-C₃)alkyl; and —SO₂(C₁-C₃)alkyl;

b is 1, 2, 3, 4 or 5; and

indicates a single bond, whereby the compounds of formula I may be ineither the E or the Z conformation;

R³ is independently selected from halogen; (C₁-C₆)alkyl; —OR⁴; —C≡N;—C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴; —(C₁-C₆)—OR⁴; nitro;phosphonato; —NHC(═O)(C₁-C₆)alkyl; sulfamyl; —OC(═O)(C₁-C₃)alkyl,preferably —OC(═O)CH₃; —O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably—O(C₂-C₆)—N(CH₃)₂; (C₁-C₃)perfluoroalkyl; and (i) or (ii) below:

wherein:

each M is a bivalent connecting group independently selected from thegroup consisting of —(C₁-C₆)alkylene-, —(CH₂)_(d)—V—(CH₂)_(e)—,—(CH₂)_(f)—W—(CH₂)_(g)— and -Z-;

each y is independently selected from the group consisting of 0 and 1;

each V is independently selected from the group consisting of arylene,heteroarylene, —C(═O)—, —C(═O)(C₁-C₆)perfluoroalkylene, —C(═O)—,—C(═S)—, —S(═O)—, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴— and —SO₂NR⁴—;

each W is independently selected from the group consisting of —NR⁴—, —O—and —S—;

each d is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each e is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each f is independently selected from the group consisting of 1, 2 and3, preferably from 1 and 2;

each g is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

wherein the absolute stereochemistry of -Z- is S or R, or a mixture of Sand R;

each R^(α) is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, —(CH₂)₃—NH—C(NH₂)(═NH), —CH₂C(═O)NH₂, —CH₂COOH, —CH₂SH,—(CH₂)₂C(═O)—NH₂, —(CH₂)₂COOH, —CH₂—(2-imidazolyl), —(CH₂)₄—NH₂,—(CH₂)₂—S—CH₃, phenyl, CH₂-phenyl, —CH₂—OH, —CH(OH)—CH₃,—CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl); and includes compounds whereinR^(α) and R⁴ combine to form a 5-, 6- or 7-membered heterocyclic orcarbocyclic ring;

a is 1, 2 or 3;

R⁴ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, substituted —(C₁-C₆)alkyl, —(C₂-C₆)alkenyl, substituted—(C₂-C₆)alkenyl and heteroalkyl wherein two R⁴ groups may together forma heterocycle;

each R⁵ is independently selected from the group consisting of —R⁴;unsubstituted aryl; substituted aryl; substituted heterocyclic;unsubstituted heterocyclic; —CO₂R⁴; —C(═O)NR⁴ ₂; —C(═NH)—NR⁴ ₂;—(C₁-C₆)perfluoroalkyl; —CF₂Cl; —P(═O)(OR⁴)₂; —OP(═O)(OR⁴)₂; —CR⁴R⁷R⁸;and a monovalent peptidyl moiety with a molecular weight of less than1000, preferably with a molecular weight of less than 800, morepreferably with a molecular weight of less than 600, most preferablywith a molecular weight of less than 400; provided that when y is 0 andR⁵ is —CO₂R⁴; then R⁴ is not —H;

each R⁶ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, and aryl(C₁-C₃)alkyl,

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, —C(═O)R⁸, —OR⁴, —SR⁴, —OC(═O)(CH₂)₂CO₂R⁶, guanidino, NR⁴₂, —NR⁴ ₃ ⁺, —N⁺(CH₂CH₂OR⁵)₃, halogen, phenyl, substituted phenyl,heterocyclyl, and substituted heterocyclyl; and

each R⁸ is independently selected from the group consisting of R^(α),halogen, —NR⁴ ₂ and heterocycles containing two nitrogen atoms;

wherein the substituents for the substituted aryl and substitutedheterocyclic groups comprising or included within Ar, R¹, R⁵ R⁶, R⁷ andR^(α) are independently selected from the group consisting of halogen;(C₁-C₆)alkyl; (C₁-C₆)alkoxy; —NO₂; —C—N; —C(═O)O(C₁-C₃)alkyl; —OR⁴;—(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;carbamyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂; and(C₁-C₃)perfluoroalkyl; or a salt of such a compound.

According to preferred embodiment of the above method of treating anindividual for a proliferative disorder, particularly cancer,

-Z- is:

wherein the absolute stereochemistry of -Z- is either S or R; and

each R^(α) is independently selected from the group consisting of —H,—CH₃, —(CH₂)₃—NH—C(NH₂)(═NH), —CH₂C(═O)NH₂, —CH₂COOH, —CH₂SH,—(CH₂)₂C(═O)—NH₂, —(CH₂)₂COOH, —CH₂-(2-imidazolyl), —CH(CH₃)—CH₂CH₃,—CH₂CH(CH₃)₂, —(CH₂)₄—NH₂, —(CH₂)₂—S—CH₃, phenyl, —CH₂-phenyl,—CH₂—CH(OH)—CH₃, —CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl), —CH(CH₃)₂ and—CH₂CH₃; and includes compounds wherein R^(α) and R⁴ combine to form a5-, 6- or 7-membered heterocyclic ring;

b is 1, 2 or 3;

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl and —(C₁-C₆)acyl;

each V is independently selected from the group consisting of —C(═O)—,—C(═S)—, —S(═O, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴— and —SO₂NR⁴—;

each R⁵ is independently selected from the group consisting of —R⁴,unsubstituted aryl, substituted aryl, substituted heterocyclic,unsubstituted heterocyclic, —CO₂R⁴, —C(═O)NR⁴ ₂, —C(═NH)—NR⁴ ₂, and amonovalent peptidyl moiety with a molecular weight of less than 1000;provided that when y is 0 and R⁵ is —CO₂R⁴; then R⁴ is not —H; and

wherein the substituents for the substituted aryl and substitutedheterocyclic groups comprising or included within Ar, R¹, R⁵ and R^(α)are independently selected from the group consisting of halogen,—(C₁-C₆)alkyl, —(C₁-C₆)alkoxy, —NO₂, —C≡N, —C(═O)O(C₁-C₃)alkyl, —OR⁴,—(C₂-C₆)—OR⁴, phosphonato, —NR⁴ ₂, —NHC(═O)(C₁-C₆)alkyl, sulfamyl,carbamyl, —OC(═O)(C₁-C₃)alkyl, —O(C₂-C₆)—N((C₁-C₆)alkyl)₂ and—(C₁-C₃)perfluoroalkyl; or a salt of such a compound.

According to another embodiment, a method of treating an individual fora proliferative disorder, particularly cancer, is provided, comprisingadministering to said individual an effective amount of at least oneconjugate of the formula I-L-Ab or Ic-L-Ab, as described herein, aloneor in combination with a pharmaceutically acceptable carrier.

According to a further embodiment of the invention, a method of inducingapoptosis of tumor cells in an individual afflicted with cancer isprovided, comprising administering to the individual an effective amountof at least one compound of formula I^(ii) as described below, alone orin combination with a pharmaceutically acceptable carrier.

According to another embodiment, a method of inducing apoptosis ofcancer cells, more preferably tumor cells, in an individual afflictedwith cancer is provided, comprising administering to said individual aneffective amount of at least one conjugate of the formula I-L-Ab, orIc-L-Ab, as described herein, alone or in combination with apharmaceutically acceptable carrier.

According to another embodiment of the invention, a method of inhibitingthe growth of tumor cells in an individual afflicted with cancer isprovided, comprising administering to the individual an effective amountof at least one compound of formula I^(ii) as described below, alone orin combination with a pharmaceutically acceptable carrier.

According to another embodiment of the invention, a method of inhibitinggrowth of tumor cells in an individual afflicted with cancer is providedcomprising administering to said individual an effective amount of atleast one conjugate of the formula I-L-Ab or Ic-L-Ab, as describedherein, alone or in combination with a pharmaceutically acceptablecarrier.

According to another embodiment of the invention, a method of reducingor eliminating the effects of ionizing radiation on normal calls in anindividual who has incurred or is at risk for incurring exposure toionizing radiation, is provided. This method comprises administering tothe individual either prior to, or after the exposure to ionizingradiation, at least one aryl or heteroaryl propene amide of formulaI^(ii) as described below, alone or in combination with apharmaceutically acceptable carrier.

According to a sub-embodiment thereof, there is provided a method ofsafely increasing the dosage of therapeutic ionizing radiation used inthe treatment of cancer or another proliferative disorder, comprisingadministering an effective amount of at least one radioprotectivecompound of formula I^(ii) as described below, alone or in combinationwith a pharmaceutically acceptable carrier. This radioprotectivecompound induces a temporary radioresistant phenotype in the normaltissue of the individual.

According to another sub-embodiment thereof, there is provided a methodfor treating a subject who has incurred, or is at risk for incurringremediable radiation damage from exposure to ionizing radiation. Thismethod comprises administering an effective amount of at least oneradioprotective compound of formula I^(ii) as described below, eitherprior to, or after the individual incurs remediable radiation damagefrom exposure to ionizing radiation.

According to another embodiment of the invention, a method of reducingor eliminating the effects of ionizing radiation on normal calls in anindividual who has incurred or is at risk for incurring exposure toionizing radiation, is provided. This method comprises administering tothe individual either prior to, or after the exposure to ionizingradiation, an effective amount of at least one conjugate of the formulaI-L-Ab or Ic-L-Ab, as described herein.

According to a sub-embodiment thereof, there is provided a method ofsafely increasing the dosage of therapeutic ionizing radiation used inthe treatment of cancer or another proliferative disorder, comprisingadministering an effective amount of at least one conjugate of theformula I-L-Ab or Ic-L-Ab, as described herein, alone or in combinationwith a pharmaceutically acceptable carrier. This radioprotectivecompound induces a temporary radioresistant phenotype in the normaltissue of the individual.

According to another sub-embodiment thereof, there is provided a methodfor treating a subject who has incurred, or is at risk for incurringremediable radiation damage from exposure to ionizing radiation. Thismethod comprises administering an effective amount of at least oneconjugate of the formula I-L-Ab or Ic-L-Ab, as described herein, eitherprior to, or after the individual incurs remediable radiation damagefrom exposure to ionizing radiation.

According to another embodiment of the invention, there is provided amethod of treating an individual for a proliferative disorder,particularly cancer, comprising:

(1) administering to the individual an effective amount of at least oneradioprotective compound of formula I^(ii) as described below; and

(2) administering an effective amount of therapeutic ionizing radiation.

According to another embodiment of the invention, there is provided amethod of treating an individual for a proliferative disorder,particularly cancer, comprising:

(1) administering to the individual administering an effective amount ofat least one conjugate of the formula I-L-Ab or Ic-L-Ab as describedherein; and

(2) administering an effective amount of therapeutic ionizing radiation.

According to another embodiment of the invention, there is provided amethod of reducing the number of malignant cells in the bone marrow ofan individual, comprising

(1) removing a portion of the individual's bone marrow,

(2) administering an effective amount of at least one radioprotectivecompound of formula I^(ii) as described below, to the removed bonemarrow;

(3) irradiating the removed bone marrow with an effective amount ofionizing radiation; and

(4) replacing the removed bone marrow with the treated bone marrow.

According to another embodiment of the invention, there is provided amethod of reducing the number of malignant cells in the bone marrow ofan individual, comprising

(1) removing a portion of the individual's bone marrow,

(2) administering an effective amount of at least one conjugate of theformula I-L-Ab or Ic-L-Ab, as described herein, to the removed bonemarrow;

(3) irradiating the removed bone marrow with an effective amount ofionizing radiation; and

(4) replacing the removed bone marrow with the treated bone marrow.

According to another embodiment of the invention, there is provided amethod for protecting an individual from cytotoxic side effects of theadministration of a cytotoxic agent, particularly a mitotic phase cellcycle inhibitor or a topoisomerase inhibitor, comprising administeringto the individual, in advance of the administration of the cytotoxicagent, an effective amount of at least one cytoprotective compound offormula I^(ii) as described below;

wherein the mitotic phase cell cycle inhibitor or topoisomeraseinhibitor is not a compound of formula I^(ii).

According to a sub-embodiment thereof, there is provided the abovedescribed method wherein the cytotoxic agent is a mitotic cell phaseinhibitor, particularly selected from vinca alkaloids, taxanes,naturally occurring macrolides, colchicine and derivatives ofcolchicine.

More particularly, the mitotic cell phase inhibitor is selected frompaclitaxel and Vincristine. Paclitaxel is an anti-mitotic drug presentlyused as an initial treatment for ovarian, breast and lung cancer, withmoderate success. Vincrisitin is a well-established anti-mitotic drugwidely used for the treatment of breast cancer, Hodgkin's lymphoma andchildhood cancers.

According to another sub-embodiment thereof, there is provided the abovedescribed method wherein the cytotoxic agent is a topoisomerase,particularly selected from the group consisting of camptothecin,etoposide and mitoxanthrone.

According to another embodiment of the invention, there is provided amethod of treating an individual for a proliferative disorder,particularly cancer, comprising:

(1) administering to the individual an effective amount of at least onecytoprotective compound of formula I^(ii) as described below, and

(2) administering an effective amount of at least one mitotic cell phaseinhibitor or topoisomerase inhibitor after administration of thecytoprotective compound of formula I^(ii).

According to another embodiment of the invention, there is provided amethod of treating an individual for a proliferative disorder,particularly cancer, comprising:

(1) administering to the individual an effective amount of at least oneconjugate of the formula I-L-Ab or Ic-L-Ab, as described herein, and

(2) administering an effective amount of at least one mitotic cell phaseinhibitor or topoisomerase inhibitor after administration of thecytoprotective compound of formula I^(ii).

For the aforementioned therapeutic methods of treatment and methods ofusing radioprotective or cytoprotective compounds of the presentinvention, the administered compound is a compound according to formulaI^(ii), wherein:

wherein:

ring A and ring B are independently selected from the group consistingof aryl and heteroaryl;

X is O or S, preferably O;

R¹ is independently selected from the group consisting of —R⁴,—SO₂(C₁-C₆)alkyl, —C(═O)R⁴, —C(═O)OR⁴, —C(═O)O(C₁—C₆)alkylenearyl, —OR⁴,—(C₂-C₆)alkynyl, —(C₃-C₆)heteroalkenyl, —(C₂-C₆)alkylene-OR⁴,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aryl(C₁-C₃)alkyl, unsubstitutedaryl(C₁-C₃)alkyl, substituted heteroaryl(C₁-C₃)alkyl and unsubstitutedheteroaryl(C₁-C₃)alkyl;

R² is independently selected from —(C₁-C₆)alkyl; halogen; —OR⁴; —C≡N;—NO₂; —CO₂R⁴; —C(═O)NR⁴ ₂; —C(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴;—(C₁-C₆)—OR⁴; phosphonato; —NR⁴ ₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;carbamyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;—S(C₁-C₃)alkyl; —S(═O)(C₁-C₃)alkyl; —(C₁-C₃)perfluoroalkyl; and—SO₂(C₁-C₃)alkyl;

b is 1, 2, 3, 4 or 5; and

indicates a single bond, whereby the compounds of formula I may be ineither the E or the Z conformation;

R³ is independently selected from halogen; —(C₁-C₆)alkyl; —OR⁴; —C≡N;—C(═O)NR⁴ ₂; —C(═O)OR⁴; —(═NR⁴)NR⁴ ₂; —O(C₁-C₃)alkylene-CO₂R⁴;—(C₁-C₆)—OR⁴; nitro; phosphonato; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;carbamyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂;—(C₁-C₃)perfluoroalkyl; and (i) or (ii) below:

wherein:

each M is a bivalent connecting group independently selected from thegroup consisting of —(C₁-C₆)alkylene-, —(CH₂)_(d)—V—(CH₂)_(e)—,—(CH₂)_(f)—W—(CH₂)_(g)— and -Z-;

each y is independently selected from the group consisting of 0 and 1;

each V is independently selected from the group consisting of arylene,heteroarylene, —C(═O)—, —C(═O)(C₁-C₆)perfluoroalkylene, —C(═O)—,—C(═S)—, —S(═O)—, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴— and —SO₂NR⁴—;

each W is independently selected from the group consisting of —NR⁴—, —O—and —S—;

each d is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each e is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

each f is independently selected from the group consisting of 1, 2 and3, preferably from 1 and 2;

each g is independently selected from the group consisting of 0, 1 and2, preferably from 0 and 1;

wherein the absolute stereochemistry of -Z- is S or R, or a mixture of Sand R;

each R^(α) is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, —(CH₂)₃—NH—C(NH₂)(═NH), —CH₂C(═O)NH₂, —CH₂COOH, —CH₂SH,—(CH₂)₂C(═O)—NH₂, —(CH₂)₂COOH, —CH₂-(2-imidazolyl), —(CH₂)₄—NH₂,—(CH₂)₂—S—CH₃, phenyl, —CH₂-phenyl, —CH₂—OH, —CH(OH)—CH₃,—CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl); and includes compounds whereinR^(α) and R⁴ combine to form a 5-, 6- or 7-membered heterocyclic orcarbocyclic ring;

a is 1, 2 or 3;

R⁴ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, substituted —(C₁-C₆)alkyl, —(C₂-C₆)alkenyl, substituted—(C₂-C₆)alkenyl and heteroalkyl wherein two R⁴ groups may together forma heterocycle;

each R⁵ is independently selected from the group consisting of —R⁴;unsubstituted aryl; substituted aryl; substituted heterocyclic;unsubstituted heterocyclic; —CO₂R⁴; —C(═O)NR⁴ ₂; —C(═NH)—NR⁴ ₂;—(C₁-C₆)perfluoroalkyl; —CF₂Cl; —P(═O)(OR⁴)₂; —OP(═O)(OR⁴)₂; —CR⁴R⁷R⁸;and a monovalent peptidyl moiety with a molecular weight of less than1000, preferably with a molecular weight of less than 800, morepreferably with a molecular weight of less than 600, most preferablywith a molecular weight of less than 400; provided that when y is 0 andR⁵ is —CO₂R⁴; then R⁴ is not —H;

each R⁶ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, and aryl(C₁-C₃)alkyl,

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl, —C(═O)R⁸, —OR⁴, —SR⁴, —OC(═O)(CH₂)₂CO₂R⁶, guanidino, —NR⁴₂, —NR⁴ ₃ ⁺, —N⁺(CH₂CH₂OR⁵)₃, halogen, phenyl, substituted phenyl,heterocyclyl, and substituted heterocyclyl; and

each R⁸ is independently selected from the group consisting of R^(α),halogen, —NR⁴ ₂ and heterocycles containing two nitrogen atoms;

wherein the substituents for the substituted aryl and substitutedheterocyclic groups comprising or included within Ar, R¹, R⁵ R⁶, R⁷ andR^(α) are independently selected from the group consisting of halogen;—(C₁-C₆)alkyl; —(C₁-C₆)alkoxy; —NO₂; —C≡N; —C(═O)O(C₁-C₃)alkyl; —OR⁴;—(C₂-C₆)—OR⁴; phosphonato; —NR⁴ ₂; —NHC(═O)(C₁-C₆)alkyl; sulfamyl;carbamyl; —OC(═O)(C₁-C₃)alkyl, preferably —OC(═O)CH₃;—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, preferably —O(C₂-C₆)—N(CH₃)₂; and—(C₁-C₃)perfluoroalkyl; or a salt of such a compound.

For the aforementioned therapeutic methods of treatment and methods ofusing radioprotective or cytoprotective compounds of the presentinvention, the administered compound is a compound according to formulaI^(ii), wherein:

According to one preferred embodiment of a compound according to formulaI^(ii),

-Z- is:

wherein the absolute stereochemistry of -Z- is either S or R; and

each R^(α) is independently selected from the group consisting of —H,—CH₃, —(CH₂)₃—NH—C(NH₂)(═NH), —CH₂C(═O)NH₂, —CH₂COOH, —CH₂SH,—(CH₂)₂C(═O)—NH₂, —(CH₂)₂COOH, —CH₂-(2-imidazolyl), —CH(CH₃)—CH₂CH₃,—CH₂CH(CH₃)₂, —(CH₂)₄—NH₂, —(CH₂)₂—S—CH₃, phenyl, —CH₂-phenyl,—CH₂—CH(OH)—CH₃, —CH₂-(3-indolyl), —CH₂-(4-hydroxyphenyl), —CH(CH₃)₂ and—CH₂CH₃; and includes compounds wherein R^(α) and R⁴ combine to form a5-, 6- or 7-membered heterocyclic ring;

b is 1, 2 or 3;

each R⁷ is independently selected from the group consisting of —H,—(C₁-C₆)alkyl and —(C₁-C₆)acyl;

each V is independently selected from the group consisting of —C(═O)—,—C(═S)—, —S(═O)—, —SO₂—, —C(═O)NR⁴—, —C(═S)NR⁴— and —SO₂NR⁴—;

each R⁵ is independently selected from the group consisting of —R⁴,unsubstituted aryl, substituted aryl, substituted heterocyclic,unsubstituted heterocyclic, —CO₂R⁴, —C(═O)NR⁴ ₂, —C(═NH)—NR⁴ ₂, and amonovalent peptidyl moiety with a molecular weight of less than 1000;provided that when y is 0 and R⁵ is —CO₂R⁴; then R⁴ is not —H; and

wherein the substituents for the substituted aryl and substitutedheterocyclic groups comprising or included within Ar, R¹, R⁵ and R^(α)are independently selected from the group consisting of halogen,—(C₁-C₆)alkyl, —(C₁-C₆)alkoxy, —NO₂, —C≡N, —C(═O)O(C₁-C₃)alkyl, —OR⁴,—(C₂-C₆)—OR⁴, phosphonato, —NR⁴ ₂, —NHC(═O)(C₁-C₆)alkyl, sulfamyl,carbamyl, —OC(═O)(C₁-C₃)alkyl, —O(C₂-C₆)—N((C₁-C₆)alkyl)₂ and—(C₁-C₃)perfluoroalkyl; or a salt of such a compound.

The propene amides of the invention are characterized by isomerismresulting from the presence of an olefinic double bond. This isomerismis commonly referred to as cis-trans isomerism, but the morecomprehensive naming convention employs E and Z designations. Thecompounds are named according to the Cahn-Ingold-Prelog system, theIUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclatureof Organic Chemistry, John Wiley & Sons, Inc., New York, NY, 4^(th) ed.,1992, p. 127-138, the entire contents of which is incorporated herein byreference. Using this system of nomenclature, the four groups about adouble bond are prioritized according to a series of rules. Then, thatisomer with the two higher ranking groups on the same side of the doublebond is designated Z (for the German word “zusammen”, meaning together).The other isomer, in which the two higher ranking groups are on oppositesides of the double bond, is designated E (for the German word“entgegen”, which means “opposite”). Both E and Z conformations areincluded in the scope of the compounds of the present invention. The Econformation is preferred.

The preferred compounds of the present invention have a particularspatial arrangement of substituents on the aromatic rings, which isrelated to the structure activity relationship demonstrated by thecompound class. Often such substitution arrangement is denoted by anumbering system, however numbering systems are often not consistentbetween different ring systems. In six-membered aromatic systems, thespatial arrangements are specified by the common nomenclature “para” for1,4-substitution, “meta” for 1,3-substitution and “ortho” for1,2-substitution as shown below.

Since aromatic rings are essentially planar, these designationsessentially define geometric positions on a six-membered ring that couldbe communicated geometrically, i.e., the ortho substituent forms aplanar angle of 60° with the substituent to which it is beingreferenced. Likewise, a meta substituent defines a 120° planar angle anda para substituent defines a 180° angle.

To designate substituent patterns in a general way for any planar ringsystem, the ortho-meta-para nomenclature is only descriptive forsix-membered monocycles, i.e., there is no “para” substituent on afive-membered aromatic ring or a bicyclic ring. However, definition of aplanar angle or a range of planar angles between two substituents is aconvention which will readily communicate a particular substitutionpattern that is independent of the particular ring involved. Thus, apara substituent in a six-membered aromatic ring is closely approximatedin other planar mono- or bicyclic rings by any substituent which, withthe reference substituent forms a planar angle of between about 144° andabout 180°. Likewise, a meta substituent in a six -membered aromaticring is approximated in other planar mono- or bicyclic rings by anysubstituent which, with the reference substituent forms a planar angleof between about 90° and about 144°. Several examples of substituentpatterns which could be communicated in this way are depicted below.

In some instances, for example, a naphthalene system substituted at the1- and 5-positions as shown in the (e) structure above, a true angle isnot formed because there is no geometric intersection between the linesdefined by the 1- and 5-position bonds. However, it is reasonable toregard these “parallel” bonds as defining a 180° angle and thusapproximating the para-arrangement of a six-membered planar ring.

The term “acyl” means a radical of the general formula —C(═O)—R, wherein—R is hydrogen, hydrocarbyl, amino or alkoxy. “Examples include forexample, acetyl (—C(═O)CH₃), propionyl (—C(═O)CH₂CH₃), benzoyl(—C(═O)C₆H₅). Phenylacetyl (—C(═O)CH₂C₆H₅), carboethoxy (—CO₂Et), anddimethylcarbamoyl (—C(═O)N(CH₃)₂).

The term “alkyl”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon radical, including di- and multi-radicals, having the numberof carbon atoms designated (i.e. C₁-C₆ means one to six carbons) andincludes straight, branched chain or cyclic groups. Examples include:methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,neopentyl, hexyl, cyclohexyl and cyclopropylmethyl. Most preferred is(C₁-C₃)alkyl, particularly ethyl, methyl and isopropyl.

The term “alkenyl” employed alone or in combination with other terms,means, unless otherwise stated, a stable monounsaturated ordi-unsaturated hydrocarbon radical straight chain, branched chain orcyclic hydrocarbon group having the stated number of carbon atoms.Examples include vinyl, propenyl (allyl), crotyl, isopentenyl,butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, cyclopentenyl,cyclopentadienyl and the higher homologs and isomers. A divalent radicalderived from an alkene is exemplified by —CH═CH—CH₂—.

Substituted alkyl or alkenyl means alkyl or alkenyl, as defined above,substituted by one, two or three substituents selected from the groupconsisting of halogen, —OH, —NH₂, —N(CH₃)₂, —CO₂H, —CO₂(C₁-C₄)alkyl,—CF₃, —CONH₂, —SO₂NH₂, —C(═NH)NH₂, —CN and —NO₂, preferably containingone or two substituents selected from halogen, —OH, NH₂, —N(CH₃)₂,trifluoromethyl and—CO₂H, more preferably selected from halogen and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

The term “alkylene”, by itself or as part of another substituent means,unless otherwise stated, a divalent straight, branched or cyclic chainhydrocarbon radical.

The term “alkoxy” employed alone or in combination with other termsmeans, unless otherwise stated, an alkyl group having the designatednumber of carbon atoms, as defined above, connected to the rest of themolecule via an oxygen atom, such as, for example, methoxy, ethoxy,1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.Preferred are (C₁-C₃)alkoxy, particularly ethoxy and methoxy.

The term “amine” or “amino” refers to radicals of the general formula—NRR′, wherein R and R′ are independently selected from hydrogen or ahydrocarbyl radical, or wherein R and R′ combined form a heterocycle.Examples of amino groups include: —NH₂, methyl amino, diethyl amino,anilino, benzyl amino, piperidinyl, piperazinyl and indolinyl.

The term “carbamyl” means the group —C(═O)NRR′, wherein R and R′ areindependently selected from hydrogen or a hydrocarbyl radical, orwherein R and R′ combined form a heterocycle. Examples of carbamylgroups include: —C(═O)NH₂ and —C(═O)N(CH₃)₂.

The term “carboxy(C₁-C₃)alkoxy” means a radical in which the carboxygroup —COOH is attached to a carbon of a straight or branched chainalkoxy group containing one to three carbon atoms. The radical thuscontains up to four carbon atoms. Examples include: —O(CH₂)₃CO₂H and—O(CH₂)₂CO₂H.

The term “cycloalkyl” refers to ring-containing alkyl radicals. Examplesinclude cyclohexyl, cyclopentyl, cyclopropyl methyl and norbomyl

The term “heteroalkyl” by itself or in combination with another termmeans, unless otherwise stated, a stable straight or branched chainradical consisting of the stated number of carbon atoms and one or twoheteroatoms selected from the group consisting of O, N, and S, andwherein the nitrogen and sulfur atoms may be optionally oxidized and thenitrogen heteroatom may be optionally quaternized. The heteroatom(s) maybe placed at any position of the heteroalkyl group, including betweenthe rest of the heteroalkyl group and the fragment to which it isattached, as well as attached to the most distal carbon atom in theheteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃, —CH₂—CH₂CH₂—OH,—CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(′O)—CH₃. Up to twoheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃, or—CH₂—CH₂—S—S—CH₃.

The term “heteroalkenyl” by itself or in combination with another termmeans, unless otherwise stated, a stable straight or branched chainmonounsaturated or di-unsaturated hydrocarbon radical consisting of thestated number of carbon atoms and one or two heteroatoms selected fromthe group consisting of O, N, and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. Up to two heteroatoms may be placedconsecutively. Examples include —CH═CH—O—CH₃, —CH═CH—CH₂—OH,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, and —CH₂—CH═CH—CH₂—SH.

The term “hydroxyalkyl” means an alkyl radical wherein one or more ofthe carbon atoms is substituted with hydroxy. Examples include—CH₂CH(OH)CH₃ and —CH₂CH₂OH.

The terms “halo” or “halogen” by themselves or as part of anothersubstituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

The term “di(C₁-C₆)alkylamino(C₂-C₆)alkoxy” or“—O(C₂-C₆)—N((C₁-C₆)alkyl)₂, means (alkyl)₂N(CH₂),O— wherein the twoalkyl chains connected to the nitrogen atom independently contain fromone to six carbon atoms, preferably from one to three carbon atoms, andz is an integer from 2 to 6. Preferably, z is 2 or 3. Most preferably, zis 2, and the alkyl groups are methyl, that is, the group is thedimethylaminoethoxy group, (CH₃)₂NCH₂CH₂O—.

The term “(C_(x)-C_(y))perfluoroalkyl,” wherein x<y, means an alkylgroup with a minimum of x carbon atoms and a maximum of y carbon atoms,wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is—(C₁-C₆)perfluoroalkyl, more preferred is —(C₁-C₃)perfluoroalkyl, mostpreferred is —CF₃.

The term “phosphonato” means the group —PO(OH)₂.

The term “sulfamyl” means the group —SO₂NRR′, wherein R and R′ areindependently selected from hydrogen or a hydrocarbyl radical, orwherein R and R′ combined form a heterocycle. Examples of sulfamylgroups include: —SO₂NH₂, —SO₂N(CH₃)₂ and —SO₂NH(C₆H₅). Preferred are—SO₂NH₂, SO₂N(CH₃)₂ and —SO₂NHCH₃.

The term “aromatic” refers to a carbocycle or heterocycle having one ormore polyunsaturated rings having aromatic character (4n+2) delocalizedπ (pi) electrons).

The term “aryl” employed alone or in combination with other terms,means, unless otherwise stated, a carbocyclic aromatic system containingone or more rings (typically one, two or three rings) wherein such ringsmay be attached together in a pendent manner, such as a biphenyl, or maybe fused, such as naphthalene. Examples include phenyl; anthracyl; andnaphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl.

The term “aryl-(C₁-C₃)alkyl” means a radical wherein a one to threecarbon alkylene chain is attached to an aryl group, e.g.,—CH₂CH₂-phenyl. Preferred is aryl(CH₂)— and aryl(CH(CH₃))—. The term“substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkyl radical inwhich the aryl group is substituted. Preferred is substitutedaryl(CH₂)—. Similarly, the term “heteroaryl(C₁-C₃)alkyl” means a radicalwherein a one to three carbon alkylene chain is attached to a heteroarylgroup, e.g., —CH₂CH₂-pyridyl. Preferred is heteroaryl(CH₂)—. The term“substituted heteroaryl-(C₁-C₃)alkyl” means a heteroaryl-(C₁-C₃)alkylradical in which the heteroaryl group is substituted. Preferred issubstituted heteroaryl(CH₂)—.

The term “arylene,” by itself or as part of another substituent means,unless otherwise stated, a divalent aryl radical. Preferred are divalentphenyl radicals, particularly 1,4-divalent phenyl radicals.

The term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself oras part of another substituent means, unless otherwise stated, anunsubstituted or substituted, stable, mono- or multicyclic heterocyclicring system which consists of carbon atoms and at least one heteroatomselected from the group consisting of N, O, and S, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen atom may be optionally quaternized. The heterocyclic system maybe attached, unless otherwise stated, at any heteroatom or carbon atomwhich affords a stable structure.

The term “heteroaryl” or “heteroaromatic” refers to a heterocycle havingaromatic character. A polycyclic heteroaryl may include one or morerings which are partially saturated. Examples includetetrahydroquinoline and 2,3-dihydrobenzofuryl. For compounds of formulaI, the attachment point on ring A or ring B is understood to be on anatom which is part of an aromatic monocyclic ring or a ring component ofa polycyclic aromatic which is itself an aromatic ring.

Examples of non-aromatic heterocycles include monocyclic groups such as:Aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

Examples of heteroaryl groups include: Pyridyl, pyrazinyl, pyrimidinyl,particularly 2- and 4-pyrimidinyl, pyridazinyl, thienyl, furyl,pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl,pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl,1,2,3-traizolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles include: Indolyl, particularly 3-,4-, 5-, 6- and 7-indolyl, indolinyl, quinolyl, tetrahydroquinolyl,isoquinolyl, particularly 1- and 5-isoquinolyl,1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl, particularly 2-and 5-quinoxalinyl, quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, benzofuryl, particularly3-, 4-, 1,5-naphthyridinyl, 5-, 6- and 7-benzofuryl,2,3-dihydrobenzofiryl, 1,2-benzisoxazolyl, benzothienyl, particularly3-, 4-, 5-, 6-and 7-benzothienyl, benzoxazolyl, benzthiazolyl,particularly 2-benzothiazolyl and 5-benzothiazolyl, purinyl,benzimidazolyl, particularly 2-benzimidazolyl, benztriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative and not limiting.

The term “heteroarylene,” by itself or as part of another substituentmeans, unless otherwise stated, a divalent heteroaryl radical. Preferredare five- or six-membered monocyclic heteroarylene. More preferred areheteroarylene moieties comprising divalent heteroaryl rings selectedfrom pyridine, piperazine, pyrimidine, pyrazine, furan, thiophene,pyrrole, thiazole, imidazole and oxazole.

For compounds of the present invention, when an aromatic orheteroaromatic ring is attached to a position and the ring comprises apolycyclic ring which is partially saturated, the attachment point onthe aromatic or heteroaromatic ring is on a ring atom of an aromaticring component of the polycyclic ring. For example on the partiallysaturated heteroaromatic ring, 1,2,3,4-tetrahydroisoquinoline,attachment points would be ring atoms at the 5-, 6-, 7- and 8-positions.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative, not limiting.

The term “hydrocarbyl” refers to any moiety comprising only hydrogen andcarbon atoms. Preferred heteroaryl groups are (C₁-C₁₂)hydrocarbyl, morepreferred are (C₁-C₇)hydrocarbyl, most preferred are benzyl and(C₁-C₆)alkyl.

The term “substituted” means that an atom or group of atoms has replacedhydrogen as the substituent attached to another group. For aryl andheteroaryl groups, the term “substituted” refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.

Where a substituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic, with straight being preferred.

The term “antibody” is intended to encompass not only intactantigen-binding immunoglobulin molecules, but also to includeantigen-binding fragments thereof such as Fab, Fab′ and F(ab′)₂fragments, or any other fragment retaining the antigen-binding abilityof an intact antibody.

The term “humanized antibody” refers to an antibody that has itscomplementary determining regions (CDR's) derived from a non-humanspecies immunoglobulin, and the remainder of the antibody moleculederived from a human immunoglobulin.

The term “chimeric antibody” means an antibody comprising a variableregion and a constant region derived from different species.

The term “humanized chimeric antibody” is means a chimeric antibody inwhich at least the constant region is human-derived.

The term “monospecific polyclonal antibody” means an antibodypreparation comprising multiple antibody species having specificity fora single antigen.

The term “monovalent peptidyl moiety” refers to a peptide radical as asubstituent on a molecule of formula I. Such a radical has a chemicalstructure that varies from the structure of the corresponding peptide inthat the structural component of the peptide, i.e., an alpha aminogroup, a sidechain amino group, an alpha carboxyl group or a sidechaincarboxyl group, will form a different functionality when bonded to themolecule of which it is to be a substituent. For example, for a peptideas shown belowH₂N-Val-Pro-Ala-COOHwhich is a substituent on a compound of formula I, the peptide iscoupled to the compound of formula I such that a carboxyl moiety of saidpeptide is coupled to a free amine moiety on the formula I compound.Elimination of H₂O results in the formation of an amide bond. As apractical result, the corresponding monovalent peptidyl substituent isshown to the left of the dotted line in the depiction below of theaforementioned peptide bonded to a compound of formula I:

The monovalent peptide moiety may be attached via either an alpha- or asidechain amino group, or an alpha or sidechain carboxyl group. Theattachment point on the peptide moiety will depend on the functionalityat the terminus of the bivalent tether group M in a manner that is knownto one of skill in the art (see the definition.

Specifically, the peptidyl moiety may be coupled to the M bivalenttether via an alpha amino or a side chain amino group when the M tetherterminates in:

—C(═O)—, —C(═S)—, —S(═O)—, or SO₂, i.e., when the variable e is zero.

Likewise, the peptidyl moiety may be coupled to the M bivalent tethervia an alpha carboxy or a side chain carboxy group when the M tetherterminates in:

—C(═O)NR⁵—, —SO₂NR⁵—, —NR⁵—, —S— or —O—, i.e., when the variable e (org) is zero.

The term “effective amount” when used to describe therapy to a patientsuffering from a proliferative disorder, refers to the amount of acompound of formula I that inhibits the growth of tumor cells oralternatively induces apoptosis of cancer cells, preferably tumor cells,resulting in a therapeutically useful and selective cytotoxic effect onproliferative cells when administered to a patient suffering from acancer or other disorder which manifests abnormal cellularproliferation.

As used herein, “ionizing radiation” is radiation of sufficient energythat, when absorbed by cells and tissues, induces formation of reactiveoxygen species and DNA damage. This type of radiation includes X-rays,gamma rays, and particle bombardment (e.g., neutron beam, electron beam,protons, mesons and others), and is used for medical testing andtreatment, scientific purposes, industrial testing, manufacturing andsterilization, weapons and weapons development, and many other uses.Radiation is typically measured in units of absorbed dose, such as therad or gray (Gy), or in units of dose equivalence, such as the rem orsievert (Sv). The relationship between these units is given below:

rad and gray (Gy) rem and sievert (Sv) 1 rad = 0.01 Gy 1 rem = 0.01 Sv

The Sv is the Gy dosage multiplied by a factor that includes tissuedamage done. For example, penetrating ionizing radiation (e.g., gammaand beta radiation) have a factor of about 1, so 1 Sv=˜1 Gy. Alpha rayshave a factor of 20, so 1 Gy of alpha radiation=20 Sv.

By “effective amount of ionizing radiation” is meant an amount ofionizing radiation effective in killing, or reducing the proliferation,of abnormally proliferating cells in a subject. As used with respect tobone marrow purging, “effective amount of ionizing radiation” means anamount of ionizing radiation effective in killing, or in reducing theproliferation, of malignant cells in a bone marrow sample removed from asubject.

By “acute exposure to ionizing radiation” or “acute dose of ionizingradiation” is meant a dose of ionizing radiation absorbed by a subjectin less than 24 hours. The acute dose may be localized, as inradiotherapy techniques, or may be absorbed by the subject's entirebody. Acute doses are typically above 10,000 millirem (0.1 Gy), but maybe lower.

By “chronic exposure to ionizing radiation” or “chronic dose of ionizingradiation” is meant a dose of ionizing radiation absorbed by a subjectover a period greater than 24 hours. The dose may be intermittent orcontinuous, and may be localized or absorbed by the subject's entirebody. Chronic doses are typically less than 10,000 millirem (0.1 Gy),but may be higher.

By “effective amount of a radioprotective compound” is meant an amountof compound effective to reduce or eliminate the toxicity associatedwith radiation in normal cells of the subject, and also to impart adirect cytotoxic effect to abnormally proliferating cells in thesubject. As used with respect to bone marrow purging, “effective amountof the radioprotective N-aryl (or N-heteroaryl) propene amide compound”means an amount of compound effective to reduce or eliminate thetoxicity associated with radiation in bone marrow removed from asubject, and also to impart a direct cytotoxic effect to malignant cellsin the bone marrow removed from the subject.

By “at risk of incurring exposure to ionizing radiation” is meant that asubject may advertently (such as by scheduled radiotherapy sessions) orinadvertently be exposed to ionizing radiation in the future.Inadvertent exposure includes accidental or unplanned environmental oroccupational exposure.

By “effective amount” of the mitotic phase cell cycle inhibitor ortopoisomerase inhibitor is meant an amount of said inhibitor effectivein killing or reducing the proliferation of cancer cells in a hostanimal.

By ”effective amount” of the cytoprotective compound is meant an amountof compound effective to reduce the toxicity of the mitotic phase cellcycle inhibitor or topoisomerase inhibitor on normal cells of theanimal.

The term “cell cycle” refers to the usual description of celldevelopment in terms of a cycle consisting of a series ofphases—interphase and M (mitotic) phase—and the subdivision ofinterphase into the times when DNA synthesis is proceeding, known as theS-phase (for synthesis phase), and the gaps that separate the S-phasefrom mitosis. G1 is the gap after mitosis but before DNA synthesisstarts, and G2 is the gap after DNA synthesis is complete before mitosisand cell division. Interphase is thus composed of successive G1, s andG2 phases, and normally comprises 90% or more of the total cell cycletime. The M phase consists of nuclear division (mitosis) and cytoplasmicdivision (cytokinesis). During the early part of the M phase, thereplicated chromosomes condense from their extended interphasecondition. The nuclear envelope breaks down, and each chromosomeundergoes movements that result in the separation of pairs of sisterchromatids as the nuclear contents are divided. Two new nuclearenvelopes then form, and the cytoplasm divides to generate two daughtercells, each with a single nucleus. This process of cytokinesisterminates the M phase and marks the beginning of the interphase of thenext cell cycle. The daughter cells resulting from completion of the Mphase begin the interphase of a new cycle.

By “mitotic phase cell cycle inhibitor” is meant a chemical agent whosemechanism of action includes inhibition of a cell's passage through anyportion of the mitotic (M) phase of the cell cycle. Such agents include,by way of example and not limitation, taxanes, such as paclitaxel andits analogs; vinca alkaloids such as vincristine and vinblastine;colchicine and its derivatives; and naturally occurring macrolides suchas rhizoxin, maytansine, ansamitocin P-3, phomopsin A, dolastatin 10 andhalichrondin B.

By “topoisomerase inhibitor” is meant a chemical agent whose mechanismof action includes interfering with the function of a topoisomerase.

The topoisomerases constitute a group of enzymes that catalyze theconversion of DNA from one topological form to another by introducingtransient breaks in one or both strands of a DNA duplex. Topologicalisomers are molecules that differ only in their state of supercoiling.Type I topoisomerase cuts one strand of DNA and relaxes negativelysupercoiled DNA, but does not act on positively supercoiled DNA. Type IItopoisomerase cuts both strands of DNA and increases the degree ofnegative supercoiling in DNA.

Thus topoisomerase inhibitors are subdivided into inhibitors oftopoisomerase I and inhibitors of topoisomerase II. Inhibitors oftopoisomerase I include, for example, adriamycin and etoposide.Inhibitors of topoisomerase II include, for example, camptothecin,irinotecan and topotecan.

The term “individual” or “subject”, includes human beings and non-humananimals. With respect to the disclosed radioprotective andcytoprotective methods, these terms refer, unless the context indicatesotherwise, to an organism that is scheduled to incur, or is at risk forincurring, or has incurred, exposure to ionizing radiation or exposureto one or more cytotoxic chemotherapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, aryl and heteroaryl propene amidesand salts thereof are believed to selectively inhibit proliferation ofcancer cells, and kill various tumor cell types without killing (or withreduced killing of) normal cells. Cells are killed at concentrationswhere normal cells may be temporarily growth-arrested but not killed.

The compounds of the invention are believed to inhibit the proliferationof tumor cells, and for some compounds, induce cell death. Cell deathresults from the induction of apoptosis. The compounds are believedeffective against a broad range of tumor types, including but notlimited to the following: ovarian cancer, breast cancer, prostatecancer, lung cancer, renal cancer, colorectal cancer, brain cancer andleukemia.

The compounds are also believed useful in the treatment of non-cancerproliferative disorders, including but not limited to the following:hemangiomatosis in newborn, secondary progressive multiple sclerosis,chronic progressive myelodegenerative disease, neurofibromatosis,ganglioneuromatosis, keloid formation, Pagets Disease of the bone,fibrocystic disease of the breast, uterine fibroids, Peyronie'sfibrosis, Dupuytren's fibrosis, restenosis and cirrhosis.

The compounds of the invention are also believed to protect normal cellsand tissues from the effects of acute and chronic exposure to ionizingradiation.

Subjects may be exposed to ionizing radiation when undergoingtherapeutic irradiation for the treatment of the above proliferativedisorders. The compounds are believed effective in protecting normalcells during therapeutic irradiation of abnormal tissues. The compoundsare also believed useful in protecting normal cells during radiationtreatment for leukemia, especially in the purging of malignant cellsfrom autologous bone marrow grafts with ionizing radiation.

According to the invention, therapeutic ionizing radiation may beadministered to a subject on any schedule and in any dose consistentwith the prescribed course of treatment, as long as the radioprotectantcompound of the invention is administered prior to the radiation. Thecourse of treatment differs from subject to subject, and those ofordinary skill in the art can readily determine the appropriate dose andschedule of therapeutic radiation in a given clinical situation.

In addition, the compounds of the present invention are believed toprotect normal cells and tissues from the effects of exposure tocytotoxic agents such as for example, mitotic phase cell cycleinhibitors and topoisomerase inhibitors.

The compounds of the present invention differ from other knowncytoprotective agents in that they not only protect normal cells, butare also operationally cytotoxic in tumor cells. In normal cell, thecytoprotective compounds of the invention induce a reversible restingstate rendering the normal cells relatively refractory to the cytotoxiceffect of mitotic phase cell cycle inhibitors and topoisomeraseinhibitors.

Normal human fibroblasts exposed to compounds of the invention in vitroare believed to exhibit transiently reduced replication rates. When thesame cells are then exposed to a mitotic phase cell cycle inhibitor suchas paclitaxel, the cells are believed to be protected from the toxiceffects of the inhibitor. The precise cytoprotective mechanism of actionof the aryl and heteroaryl propene amides on normal tissues is unknown.However, based on experimental models, and without wishing to be boundby any theory, these compounds may affect several elements in normalcells inducing a reversible quiescent cell-cycling state in whichtransit through mitosis, and many of the changes necessary for suchpassage, are down regulated, inactivated or absent. Tumor cells appearto be refractory to this effect of the compounds and in fact continuecycling with readily activated programmed cell death pathways. Accordingto other possible mechanisms of protection, anticancer agent-inducedproinflammatory cytokine release from monocytes or macrophages,activation of JNK-1 death pathway induction, and P34Cdc2 kinase may berendered innocuous by pre-exposure to compounds of the invention.

In one embodiment of the invention, there is provided a compoundaccording to formula Ib:

wherein:

R¹ is —H;

X is O;

q is 0;

n is 0 or 1, and

the conformation of the olefin double bond is E; and

wherein R², R^(2o), R^(3m), and R^(3p) are as defined for formula Iabove.

In a further sub-embodiment of the above described compounds, there isprovided a compound,

wherein:

R^(2o) is —(C₁-C₆)alkoxy;

R² is selected from the group consisting of halogen, —(C₁-C₆)alkyl,—(C₁-C₆)alkoxy and —NR⁴ ₂; and

n is 0 or 1;

or a salt of such a compound.

In another embodiment of the invention there is provided a compound offormula Ic:

wherein:

R², R^(2o), R^(3m), R^(3p) and n, M, y and R⁶ are as defined as forformula I above; or a salt of such a compound.

In a sub-embodiment of the compounds of the invention, there is provideda compound of formula Ic,

wherein:

q is 0;

n is 1;

R³, is halogen or —(C₁-C₆)alkoxy;

R^(2o) is —(C₁-C₆)alkoxy; and

R² is —(C₁-C₆)alkoxy;

or a salt of such a compound.

In a further sub-embodiment of the compounds of the invention, there isprovided a compound of formula Ic,

wherein:

q is 0;

n is 1;

R³

l is halogen or methoxy;

R^(2o) is methoxy; and

R² is methoxy;

or a salt of such a compound.

Such compounds include, for example,(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide,and salts thereof.

The amino group of amino-substituted aryl and heteroaryl propene amidessuch as, for example,(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propen-amidemay be derivatized in several ways to form additional compounds of theinvention.

Such compounds of the invention include, for example:

-   -   2-[({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)sulfonyl]acetic        acid;    -   2-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)acetic        acid;    -   (2E)-N-[3-(amidinoamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   2-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)acetic        acid;    -   (2E)-N-{3-[(3,5-dinitrophenyl)carbonylamino]-4-methoxyphenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{3-[(3,5-diaminophenyl)carbonylamino]-4-methoxyphenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[³-(2-chloroacetylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{4-methoxy-3-[2-(4-methylpiperazinyl)acetylamino]-phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[4-methoxy-3-(phenylcarbonylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{4-methoxy-3-[(4-nitrophenyl)carbonylamino]phenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{3-[(4-aminophenyl)carbonylamino]-4-methoxyphenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{3-[(1Z)-1-aza-2-(4-nitrophenyl)vinyl]4-methoxyphenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-        ((2R)-2,6-diaminohexanoylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-((2R)-2-amino-3-hydroxypropanoylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-((2S)-2-amino-3-hydroxypropanoylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-        (aminocarbonylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[4-methoxy-3-(methylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-(acetylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-(3-{[(2,4-dinitrophenyl)sulfonyl]amino}-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-(3-{[(2,4-diaminophenyl)sulfonyl]amino}-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{3-[2-(dimethylamino)acetylamino]-4-methoxyphenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   2-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)propanoic        acid;    -   (2E)-N-(4-methoxy-3-{[4-(4-methylpiperazinyl)phenyl]-carbonylamino}phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-(2-hydroxyacetylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[4-methoxy-3-(2-pyridylacetylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)methyl        acetate;    -   (2E)-N-[3-(2-hydroxypropanoylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-{4-methoxy-3-[2-(triethylammonium)acetylamino]-phenyl}-3-        (2,4,6-trimethoxyphenyl)-prop-2-enamide;    -   (2E)-N-(4-methoxy-3-{2-[tris(2-hydroxyethyl)ammonium]-acetyl-amino}phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-[3-(2-hydroxy-2-methylpropanoylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   1-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)-isopropyl        acetate;    -   (2E)-N-[4-methoxy-3-(2,2,2-trifluoroacetylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (2E)-N-(4-methoxy-3-{[(trifluoromethyl)sulfonyl]-amino}phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   3-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)propanoic        acid;    -   3-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)propanoyl        chloride;    -   3-{[(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)methyl]oxycarbonyl}propanoic        acid;    -   4-(N-{5-[(2E)-3-(2,4,6-trirethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)butanoic        acid;    -   (2E)-N-{4-methoxy-3-[2-(phosphonooxy)acetylamino]phenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide,        disodium salt;    -   4-({5-[(2E)-3-(2,4,6-tiimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)butanoic        acid;    -   3-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)propanoic        acid;    -   (2E)-N-[4-methoxy-3-(methoxycarbonylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide,    -   (2E)-N-(4-methoxy-3-{[(4-methoxyphenyl)sulfonyl]amino}-phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   (N-{5-[(2E)-3-        (2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)ethyl        acetate;    -   methyl        3-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)propanoate;    -   ethyl        2-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)acetate;    -   (2E)-N-[4-methoxy-3-(2,2,3,3,3-pentafluoropropanoylamino)-phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   methyl        2-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoyl-amino]-2-methoxyphenyl}carbamoyl)-2,2-difluoroacetate;    -   3-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)-2,2,3,3-tetrafluoropropanoic        acid;    -   (2E)-N-[3-(2-aminoacetylamino)-4-methoxyphenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;    -   2-(N-{5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}carbamoyl)-2,2-difluoroacetic        acid;

(2E)-N-{3-[2-(dimethylamino)-2,2-difluoroacetylamino]-4-methoxy-phenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;and salts of such compounds.

The general synthesis scheme may be used in two different synthesisstrategies to produce compounds derivatized at the aromatic amino group.

The compounds of the invention may be prepared by one of severalmethods. In the synthesis methods to follow, reference to “Ar” and tothe term “aryl” is intended to include substituted and unsubstitutedaryl, and also substituted and unsubstituted heteroaryl.

According to General Procedure 1, aryl propene amides are prepared by anovel synthesis, which is depicted in Scheme 1. By this synthetic route,the aryl propene amide, I is assembled via the condensation of anintermediate aromatic malonamide acid IV with an aromatic aldehyde V.

The intermediate aromatic malonamide acid IV is prepared by hydrolysisof the corresponding aromatic malonamide ester III. This hydrolysis istypically performed at elevated temperature in using at least oneequivalent of a strong base such as, for example sodium hydroxide orlithium hydroxide. The latter reaction is conducted in a mixed aqueousand organic solvent, the organic solvent being selected from watermiscible solvents such as, for example, methanol, ethanol or THF. Theintermediate aromatic malonamide ester III is prepared by coupling anaromatic amine H with an alkylmalonyl halide. Several of these alkylmalonyl halide reagents are available commercially, including, forexample, ethyl-3-chloro-3-oxoproprionate (commonly called ethyl malonylchloride)[36239-09-5) and methyl 3-chloro-3-oxoproprionate (commonlycalled methyl malonyl chloride)[37517-81-01 (both available from AldrichChemicals). In addition these malonyl chlorides may be synthesized usingknown methods.

General Procedure 1 Step A: Synthesis of anAlkyl-2(N-Arylaminocarbonyl)acetate

According to Scheme 1, to a solution of an aromatic amine II (10 mmol)and TEA (10 mmol) in dichloromethane(DCM) (50 mL) at room temperature isslowly added a solution of an alkyl malonyl chloride (10 mmol) indichloromethane. The reaction is stirred for 1 hour. The reactionmixture is filtered and solvent is removed under reduced pressure toyield an oily material.

The crude product is purified by column chromatography to yield analkyl-2-(N -arylaminocarbonyl)-acetate III.

Step B: Synthesis of 3-Arylamino-3-oxopropanoic acid

The alkyl-2-(N-arylaminocarbonyl)-acetate III is refluxed for 2.5 hoursin a solution of sodium hydroxide (9.0 g) in water (90 mL) and ethanol(90 mL). The reaction mixture is subsequently cooled and acidified withHCl to precipitate the crude product acid. The crude3-arylamino-3-oxopropanoic acid IV is removed by filtration andrecrystallized from hot water.

Step C: Condensation of a 3-arylamino-3-oxopropanoic acid (IV) with anaromatic aldehyde (V)

A solution of the arylamino-3-oxopropanoic acid IV (10 mmol), anaromatic aldehyde V (10 mmol) and benzylamine (0.4 mL) is refluxed for 3hours in glacial acetic acid (10 mL). The solution is then cooled. Coldether (50 mL) is added. The organic layer is separated and washed with asaturated solution of sodium bicarbonate (30 mL), sodium bisulfite (30mL) and dilute hydrochloric acid (30 mL). The ether solution is thendried over anhydrous sodium sulfate and evaporated under reducedpressure to yield the corresponding N-aryl-3-aryl-2-propenamide I(E-isomer).

According to General procedure 2, which is depicted in Scheme 2, acondensation according to the method of Nodia et al, (U.S. Pat. No.4,337,270) is utilized, relying on the condensation of an intermediateE- or Z-aromatic acryloylhalide VII, such as for example, a cinnamoylchloride with an appropriate aromatic amine II, such as for example ananiline. The latter reaction is conducted in a nonprotic solvent in thepresence of a base. The same compound may serve as both the nonproticsolvent and the base. Such dual-function solvents include, for example,pyridine, substituted pyridines, trimethylamine, TEA and DIPEA. Theentire disclosure of U.S. Pat. No. 4,337,270 is incorporated herein byreference.

The intermediate E- or Z-aromatic acryloylhalide VII is prepared fromthe corresponding aromatic acrylic acid VI. The aromatic acrylic acid isreacted with a halogenating agent such as for example, thionyl chlorideor phosphorous pentachloride to form the intermediate carboxylic acidchloride VII.

General Procedure 2

Condensation of (E) or (Z)- Aromatic Acryloyl Chlorides with AromaticAmines:

According to Scheme 2, a solution of aromatic amine II (10 mmol) inpyridine (75 mL) is reacted with an (E) or (Z)-aromatic acryloyl halideVII (10 mmol) for 4 to 6 hours at 80° C. The reaction mixture is cooledand poured into ice water (IL) and concentrated hydrochloric acid (100mL) is added. The precipitated product is separated by filtration andcrystallized to yield a pure N-aryl-3-aryl-2-propenamide I. Thissynthesis is depicted in Scheme 2 below.

According to General Procedure 3, the aromatic acrylamide I is preparedby coupling the corresponding aromatic acrylic acid VI with an aromaticamine II using an amide coupling reagent such as HATU, DIC or DCC.

Aromatic acrylic acids of general formula VI are syntheticallyaccessible. The trans- or E-aromatic acrylic acids are easily preparedvia the Heck reaction (U.S. Pat. No. 3,988,358) wherein palladiumcatalysis allows reaction of an alkyl acrylate ester with an iodo orbromo-substituted aromatic ring. A similar coupling reaction may beuseful in the preparation of the cis- or Z-aromatic propene amides,wherein an isolable stannane acrylate is prepared from a 2-bromoacrylateester of the Z-conformation. (Organic Reactions, Volume 50, “The StilleReaction”, 1997) The stannane reagent should retain the Z conformationand couple the acrylate moiety to a bromo, iodo orpseudohalogen-substituted aromatic ring with retention of theZ-stereochemistry.

General Procedure 3

Condensation of (E) or (Z)-Aromatic Acrylic Acids with Aromatic Amines:

According to Scheme 3, to a solution of an aromatic acrylic acid VI (10mmol) in DCM (10 mL), is added an aromatic amine II (10 mmol) and DIPEA(5 mmol). This mixture is stirred at room temperature for 24 hours. Thereaction progress is monitored by TLC. After the reaction is complete,DCM (25 mL) is added to the reaction mixture, and the organic layer isseparated and washed with saturated sodium bicarbonate (15 mL), diluteHCl (15 mL) and water (30 mL). The organic layer is then dried(magnesium sulfate) and concentrated under reduced pressure to yield thecrude N-aryl-3-arylprop-2-enamide I, which is subsequently purified byrecrystallization.

Some functional groups on the aromatic rings, in particular aromaticamine nitrogens are further derivatizeable. Derivatives of aromaticamino groups which are useful in the present invention include, forexample: acylation to form carboxamide, carbamate and urea derivatives;sulfonylation to form sulfonamides, sulfonyl ureas and sulfamoyl esters;imine formation for formation of imines and for alkylation or arylation(or heteroarylation) via reductive amination; alkylation to form mono-or di-alkylamino derivatives, palladium catalyzed cross coupling to formN-aryl (or N-heteroaryl) derivatives by coupling with aromatic halidesor aromatic pseudo halides such as aromatic triflates. Derivatives mayalso include conjugates to biological molecules such as antibodies toyield macro molecules capable of being directed to a desired site ofaction thereby reducing or precluding side effects associated withinteraction of a drug prepared from a compound of the present inventionwith tissues and cells which are not proliferating abnormally.

The synthetic schemes shown above depict three synthetic strategies forpreparing aryl propene amides of the invention. In addition to theamides prepared in Schemes 1-3 above, compounds of the present inventioninclude thioamides of formula I wherein X is S. Thioamides of thepresent invention may be prepared according to procedures referenced inFieser 13, 38; 15, 37 and 16, 37, by reacting the amide compounds offormula I with2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide(Lawesson's reagent) according to Scheme 4, below.

The synthetic Schemes 1-3 shown above reflect a convergent synthesisstrategy. Thus A-ring and B-ring components may be synthesized andelaborated separately prior to coupling to form the target aryl propeneamides. The convergent synthetic schemes shown above allow forarrangement of the assembly steps of the aryl propene amide backbone andof derivatization of aromatic amine substituents (or other derivatizablefunctionalities) to accommodate functional group sensitivity and/or toallow for diversity elements arising from derivatization of aromaticamines to be introduced either before or after the assembly of the arylpropene amide backbone via the coupling reactions of Schemes 1-3.

To synthesize a derivative of an A-ring amine substituent on a compoundof formula I, the starting aromatic amine component II would have twoamine substituents of approximately equal reactivity. Therefore, one ofthe amine substituents must be protected with a protecting group orotherwise rendered unreactive for either the amide coupling to make thepropene amide backbone, or for the derivatization of the aminesubstituent. One approach to this differentiation of two aromatic aminegroups is to replace one of the amine groups with a nitro. The nitrogroup performs the same function as a protecting group in this synthesisbecause it “masks” the reactive amino group during a first reaction, andon reduction to the amine, can participate in the second reaction. Morespecifically, the nitro group is unreactive to the amide coupling and isgenerally unreactive to all of the amine derivatizations desired forsynthesis of compounds of the invention. Of equal importance, the nitrogroup can readily be reduced to generate an aromatic amine when needed.Reduction of the aromatic nitro group can be done, for example, viacatalytic hydrogenation. Catalytic hydrogenation provides the capabilityto selectively reduce the aromatic nitro group without reducing theolefin or other functionality in the intermediate.

A simple illustration of the flexibility of the assembly andderivatization of an aromatic amino substituent on the A-ring of acompound of formula I is depicted in Scheme 5, in which a3-acetamido-4-methoxy derivative is prepared via both strategies. InScheme 5, the first starting A-ring synthon is 3-nitro-4-methoxy aniline[577-72-0]. This starting material allows for (step 1a) coupling to formthe propene amide; (step 2a) reduction of the 3-nitro group and (step3a) acetylation of the 3-amino group.

The second starting material is 2-methoxy-5-nitroaniline [99-59-2]. Thisstarting material allows for (step 1b) acetylation of the 3-amino group;(step 2b) reduction of the 3-nitro group to the amino; and (step 3b)coupling to form the propene amide. This dual strategy is useful toafford facile and efficient diversification of either the B-ring arylpropene while keeping an elaborated A-ring aniline constant, or makingnumerous derivatives of an A-ring amine substituent as a final stepwhile keeping the B-ring aryl propene constant. The dual synthesisroutes depicted in Scheme 5 show an acetylation reaction forillustration purposes. Each of the two synthesis routes will allow for abroad range of derivatization of an amino substitution of ring A ofcompounds of formula I. In addition, though the scheme is shownspecifically for 2-methoxy-5-nitroaniline [99-59-2] and3-nitro-4-methoxy aniline [577-72-0], the aromatic amine derivatizationsand synthetic routes may be employed with starting materials of formulaSM-1 and SM-2 below,

wherein in intermediate SM-1, R^(3m) is —NO₂ and in SM-2, R^(3m) is —NH₂(i.e. R^(3m) is formula (i), m is 0, R⁴ is —H and R⁵ is —R⁴═—H).

Therefore, according to one embodiment of the invention, a process isprovided for preparing a compound of formula Ic:

or a salt thereof, comprising:

reacting an aromatic amino compound of formula Id

with an electrophilic compound of formula VIII:R⁵-L   VIII

wherein R⁵ comprises an electrophilic reactive center selected from thegroup consisting of:

(a) an alkyl moiety having a leaving group;

(b) an aryl or heteroaryl halide or aryl or heteroaryl pseudo halide;

(c) a carboxylic acid activated with a leaving group;

(d) a sulfonic acid activated with a leaving group;

(e) a carbamic acid moiety activated with a leaving group;

(f) a cyanate moiety;

(g) an aldehyde or ketone moiety, or a hydrate thereof or a ketal oracetal thereof;

(h) a carboxylic acid moiety and an amide coupling reagent; or

(i) the intermediate product of a thiourea moiety and 2-chloro-1-methylpyridinium iodide; to form a compound of formula Ic.

Thus, Table 4 below shows examples of the derivatizations which may bemade of a compound of the present invention which has an aminosubstituent on the A ring.

TABLE 4

Reagents Products Activated (A) carboxylic or (B)carbamic acids;includingcarboxylic acids or amino acidsin combination withamidecoupling reagents, to formcompounds of formula A1 andB1respectively.

Activated (C) sulfonic or (D)sulfamic acids formingcompounds of formulaC1 andD1 respectively.

Aryl or heteroaryl halide orpseudohalide (E) with Pd or Nicatalyst,forming compounds offormula E1.

Alkyl moiety with a leavinggroup (F), forming alkyl amineF1

Aldehyde or ketone moiety (G):A. formation of substitutedimine G1.B.reductive amination to formsubstituted amine G2.

Reagent product of a substitutedthiourea and a 1-methylor1-phenyl-2-halopyridinium salt,(H) reacting to form theguanidinederivative H1

For product example formulas shown in Table 4 above, the B-ring portionof the molecule, shown in Formula Id remains unchanged under thereaction conditions. Also functional groups designated by R², R^(2o),R^(3m), R^(3p), n and q are as defined for Formula Id above. Thechemistry depicted in Table 4 above is intended to be exemplary and notexclusive of additional derivatizations of aromatic amines of formula Idor of derivatizations of other aromatic amines of formula I. Inaddition, the derivatizations shown above may be done on intermediatesof formula SM-1

Reaction of compounds of formula I or Id with unactivated carboxylicacids of formula A to form carboxamides may be done for example atambient temperature using a coupling agent such as, for example DIC.Suitable solvents include for example polar aprotic solvents such asdimethyl formamide (MF). The reaction also incorporates an acidscavenger such as for example a tertiary amine to consume acid formed inthe reaction. This reaction may also be performed using reagents onsolid support, such as for example a carbodiimide bonded to apolystyrene bead. The tertiary amine acid scavenger may likewise be apolystyrene bead-bound reagent.

The carboxylic acid used in the above reaction may be an amino acid (anatural amino acid, a synthetic amino acid or a peptide fragment. Aminoacid reagents in the above reaction will be protected at the alphaamino, sidechain amino, sidechain carboxy or any other functionalitywhich would not be stable to the coupling reaction conditions. Suitableamine protecting groups include benzyl, CBZ and FMOC.

Reaction of compounds of formula I or Id with activated carboxylic acidor carbamic acid functionalities of formula A to form amides or formulaA1 and ureas of formula B1 respectively, may be done, for example atambient temperature. Activation of carbamic or carboxylic acids is byreplacement of the —OH group of a carboxy with a leaving group such as ahalide. This preparation may be done, for example by halogenating thecarboxylic or carbamic acid. A common reagent for performing thisreaction is thionyl chloride for synthesis of acid chlorides orcarbamoyl chlorides. Bromides and fluorides may also be prepared usingsuch reagents as thionyl bromide or hexafluoro acetone respectively.Other leaving groups include mixed anhydrides which may be prepared bychemistry known in the art.

Similarly, sulfonamides of formula Cl may be prepared by reactingactivated sulfonic acids formula C, particularly sulfonyl chlorides withcompounds of formula I, Id or SM-1. The reaction may be done at ambienttemperature using an acid scavenger as described above.

Reaction of compounds of formula I, Id or SM-1 with aryl or heteroarylhalides of formula E to make the corresponding diaryl amine of formulaE1, may be done using a transition metal catalyst, particularly apalladium catalyst such as for example tetrakistriphenylphosphinopalladium. The aromatic halogen compound is preferablyan aromatic bromide, however iodides and chlorides are also useful. Thereaction may be done in a dry solvent including for example suchsolvents as toluene, THF and DMF. The reaction is done in the presenceof a base such as for example cesium carbonate or potassiumtert-butoxide.

Reaction of compounds of formula I, Id or SM-1 with aldehydes or ketonesof formula G to form the imine of formula GI may be done using acidcatalysis and using methods that remove water as it is formed in thereaction. A suitable acid catalyst is toluene sulfonic acid. Suitableconditions for removing water formed in the reaction include molecularsieves and azeotropic removal via performance of the reaction in asolvent that forms a low-boiling azeotrope with water. Such solventsinclude toluene and chloroform.

In a variation of the imine formation, a reductive amination may be doneas an alternative method of preparing alkyl derivatives of the formula Iand Id aromatic amines of formula G2. In this procedure, a reducingagent is added to the reaction mixture with the aromatic amine offormula I or Id and the aldehyde or ketone. Suitable reducing conditionswill favor reduction of the product imine selectively over the startingaldehyde or ketone. Suitable conditions include for example, reductionwith sodium cyanoborohydride under acidic conditions.

Reaction of aromatic amines of formula I, Id or SM-1 to form guanidinederivatives of formula H1 may be done via reaction with a reagent H,formed by reacting a thiourea, such asN,N′-bis-(tert-butoxycarbonyl)thiourea with a 1-methyl or1-phenyl-2-halopyridinium salt, particularly 2-chloro-1-methylpyridiniumiodide.

Thus in other embodiments of the present invention, there are providedcompounds of formula A1, formula B1, formula C1, formula D1, formula E1,formula F1, formula G1, formula G2 and formula H1, wherein:

R², R^(2o), n, R^(3p), R^(3m) and q are as defined for formula Id above;

or salts of such compounds.

Preparation of Id as an Intermediate for Derivatization of the 3-AminoGroup

In a further embodiment thereof, there is provided a process forpreparing a compound of formula Id or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p) and n are defined as for formula Iabove; comprising:

chemically reducing a compound of formula Ie (E conformation):

to form a compound of formula Id or a salt of such a compound.Preparation of the 3-Nitro Intermediate Ie by Three Different RoutesA. Preparation of Ie Via Coupling an Aromatic Aldehyde with a MalonylAnilide

In a further embodiment thereof, there is provided a process forpreparing a compound of formula Ie or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q and n are defined as for formula Iabove; and wherein the olefin double bond is in the E conformation;comprising:

(1) coupling a compound of formula IIb:

with an alkyl ester of a malonic acid halide:

to yield a carboxylic ester compound of formula IIIb:

(2) hydrolyzing the carboxylic ester compound of formula IIIb to form acarboxylic acid compound of formula IVb; and

(3) coupling of the carboxylic acid compound of formula IVb with anaromatic aldehyde of formula V:

in an acidic solvent or solvent mixture, particularly, glacial aceticacid at elevated temperature to form a compound of formula Ie or a saltof such a compound.

The alkyl ester of a malonic acid halide employed as a reagent in step 1of the above preparation of a compound of formula Ic preferablycomprises a (C₁-C₁₀)alkyl ester, more preferably a (C₁-C₅)alkyl ester,most preferably a commercially available reagent such as for example themethyl or ethyl ester of malonic acid chloride.

B. Preparation of Ie Via Coupling an Aromatic Amine with an Aryl PropeneAcid Halide.

In another embodiment thereof, there is provided a process for preparinga compound of formula Ie or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q and n are defined as for formula Iabove; comprising:

(1) halogenating a carboxylic acid of formula VIb with a halogenatingagent:

to form an acid halide of formula VIIb:

(2) coupling the acid halide VIIb to an aromatic amino compound offormula IIb

to form an amide compound of formula Ie or a salt of such a compound.C. Preparation of Ie Via Coupling an Aromatic Amine with an Aryl PropeneAcid.

In another embodiment of the invention, there is provided a process forpreparing a compound of formula Ie or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q and n are defined as for formula Iabove; comprising:

reacting an aromatic amino compound of formula IIb

with a carboxylic acid compound of formula VIb:

and an amide coupling agent, to form a compound of formula Ie or a saltof such a compound.

Preparation of a Compound of Formula Ic Via Derivatization of a NitroAniline Followed by Reduction of the Nitro Group to an Amino Group andCoupling to an Aryl Propene Acid.

In another embodiment of the invention, there is provided a process forpreparing a compound of formula Ic or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q, n, M, y and R⁵ are defined as forformula I above; comprising

(1) reacting an aromatic amine of formula IX

with an electrophilic compound of formula VIII:R⁵-L   VIII

wherein L comprises an electrophilic reactive center selected from thegroup consisting of:

(a) an alkyl moiety having a leaving group;

(b) an aryl or heteroaryl halide or aryl or heteroaryl pseudo halide;

(c) a carboxylic acid activated with a leaving group;

(d) a sulfonic acid activated with a leaving group;

(e) a carbamic acid moiety activated with a leaving group;

(f) a cyanate moiety;

(g) an aldehyde or ketone moiety, or a hydrate thereof or a ketal oracetal thereof;

(h) a carboxylic acid moiety and an amide coupling reagent; or

(i) the intermediate product of a thiourea moiety and 2-chloro-1-methylpyridinium iodide; to form a compound of formula IXa:

(2) optionally protecting the —NH-(M)_(y)-R⁵ moiety;

(3) chemically reducing said nitro compound of formula Ixa to form thearomatic amine IXb:

(3) reacting aromatic amine IXb with a carboxylic acid compound offormula VIb:

and an amide coupling agent; and

(5) optionally removing said protecting group to form a compound offormula Ic; or a salt of such a compound.

The —NH-(M)_(y)-R⁵ moiety is optionally protected by a suitableprotecting group bonded to the nitrogen atom of the —NH-(M)_(y)-R⁵moiety. A suitable protecting group is one which prevents the nitrogenof the —NH-(M)_(y)-R⁵ moiety from reacting under the reaction conditionsof succeeding steps 3 and 4. Suitable protecting groups include, forexample, benzyl and substituted benzyl, CBZ, BOC and FMOC groups.

Preparation of a Compound of Formula Ic Via Coupling a 3-Amino MalonylAnilide (Derivatized at the 3-Amino) with an Aromatic Aldehyde

In a further embodiment of the invention, there is provided a processfor preparing a compound of formula Ic or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q, n, M, y and R⁵ are defined as forformula I above; and

wherein the olefin double bond is in the E conformation; comprising:

(1) coupling a compound of formula IXb:

wherein the —NH-(M)_(y)-R⁵ moiety is optionally protected with aprotecting group;

with an alkyl ester of a malonic acid halide:

to yield a carboxylic ester compound of formula IXc:

(2) hydrolyzing of the carboxylic ester compound of formula IXc to forma carboxylic acid compound of formula IXd;

(3) coupling of the carboxylic acid compound of formula IVb with anaromatic aldehyde of formula Vb:

in an acidic solvent or an acidic solvent mixture, particularly glacialacetic acid at elevated temperature; and

(4) optionally removing said protecting group to form a compound offormula Ic; or a salt of such a compound.

The —NH-(M)_(y)-R⁵ moiety is optionally protected as described in theabove preparation of a compound of formula Ic via derivatization of anitro aniline followed by reduction of the nitro group to an amino groupand coupling to an aryl propene acid.

The alkyl ester of a malonic acid halide employed as a reagent in step 1of the above preparation of a compound of formula Ic preferablycomprises a (C₁-C₁₀)alkyl ester, more preferably a (C₁-C₅)alkyl ester,most preferably a commercially available reagent such as for example themethyl or ethyl ester of malonic acid chloride.

Preparation of a Compound of Formula Ic Via Coupling a 3-Amino Aniline(Optionally Protected) with an Aryl Propene Acid Halide

In another embodiment thereof, there is provided a process for preparinga compound of formula Ic or a salt thereof:

wherein R², R^(2o), R^(3m), R^(3p), q, n, M, y and R⁵ are defined as forformula I above:

comprising:

(1) halogenating a carboxylic acid of formula VIb with a halogenatingagent:

to form an acid halide of formula VIIb:

(2) coupling the acid halide VIIb to an aromatic amino compound offormula IXb

wherein the —NH-(M)_(y)-R⁵ moiety is optionally protected with aprotecting group; and

(3) optionally removing said protecting group to form an amide compoundof formula Ic; or a salt of such a compound.

The —NH-(M)_(y)-R⁵ moiety is optionally protected as described in thetwo above preparations of a compound of formula Ic.

Preparation of Thioamide Compounds of Formula Is

In another embodiment of the invention, there is provided a process forpreparing a compound of formula Is or a salt thereof,

comprising:

reacting a compound of formula Ic:

wherein R², R^(2o), R^(3m), R^(3p), q, n, M, y and R⁵ are defined as forformula I above;

with2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide,

to form a compound of formula Is.

Halides which may comprise a leaving group component of theelectrophilic functionality R⁵-L are preferably chloro, bromo or iodo.Suitable leaving groups also include sulfonate esters such as tosylate,nosylate, mesylate and triflate. The term “pseudo halide” refers to amoiety, such as a triflate or mesylate, which behaves like a halide inpalladium or nickel-catalyzed amination reactions.

Carboxylic acid moieties which may comprise the electrophilicfunctionality R⁶-L include, for example, amino acid residues bearingoptional protecting groups on any alpha-amino functionality, sidechainamino functionality, alpha carboxylic acid functionality, sidechaincarboxylic acid functionality or other sidechain functionalities thatrequire a protecting group. Such amino acids may be naturally occurringamino acids or synthetic amino acids including amino acids of either R-or S-absolute configuration.

Following the aforesaid processes of coupling compounds: of formula IXbto compounds of formulae VIb or VIIb, of formula IXd to compounds offormula Vb, or of compounds of formula Id to compounds of formula VIII,any protecting groups used in the synthesis of a compound of formula Iare optionally removed.

The compounds of the present invention may take the form of salts. Theterm “salts”, embraces salts commonly used to form alkali metal saltsand to form addition salts of free acids or free bases. The term“pharmaceutically-acceptable salt” refers to salts which possesstoxicity profiles within a range so as to have utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility in asynthetic process. Suitable pharmaceutically-acceptable acid additionsalts may be prepared from an inorganic acid or from an organic acid.Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, example of which are formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, beta-hydroxybutyric,salicyclic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include perchloratesand tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include for example, metallic salts made from calcium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanqlamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples ofpharmaceutically unacceptable salts include lithium salts and cyanatesalts. All of these salts may be prepared by conventional means from thecorresponding aryl or heteroaryl propene amide by reacting, for example,the appropriate acid or base with the compound of formula I.

It will be understood that when compounds of the present inventioncontain one or more chiral centers, the compounds may exist in, and maybe isolated as pure enantiomeric or diastereomeric forms or as racemicmixtures. The present invention therefore includes any possibleenantiqmers, diastereomers, racemates or mixtures thereof of thecompounds of the invention which are biologically active in thetreatment of cancer or other proliferative disease states.

It is understood that due to chemical properties (i.e., resonancelending some double bond character to the C—N bond) of restrictedrotation about the amide bond linkage (as illustrated below) it ispossible, to observe separate rotamer species and even, under somecircumstances, to isolate such species. It is further understood thatcertain structural elements, including steric bulk or substituents onthe amide nitrogen, may enhance the stability of a rotamer to the extentthat a compound may be isolated as, and exist indefinitely as a singlestable rotamer. The present invention therefore includes any possiblestable rotamers of formula I which are biologically active in thetreatment of cancer or other proliferative disease states.

The compounds of the invention may be administered to individuals(mammals, including animals and humans) afflicted with cancer.

The compounds are also believed useful in the treatment of non-cancerproliferative disorders, that is, proliferative disorders which arecharacterized by benign indications. Such disorders may also be known as“cytoproliferative” or“hyperproliferative” in that cells are made by thebody at an atypically elevated rate. Such disorders include, but are notlimited to, the following: hemangiomatosis in new born, secondaryprogressive multiple sclerosis, chronic progressive myelodegenerativedisease, neurofibromatosis, ganglioneuromatosis, keloid formation,Paget's disease of the bone, fibrocystic disease of the breast, uterinefibroids, Peyronie's fibrosis, Dupuytren's fibrosis, restenosis andcirrhosis.

The compounds of the invention are further believed useful in theprotection of normal cells from the cytotoxic and genetic effects ofexposure to radiation, in individuals who have incurred, who will in thefuture incur and who are at risk for incurring exposure to ionizingradiation.

In addition, the compounds of the invention are believed useful inprotecting individuals from the cytotoxic side effects ofchemotherapeutic agents, particularly mitotic phase cell cycleinhibitors and topoisomerase inhibitors, used in the treatment of cancerand other proliferative disorders.

For treating proliferative disorders, for providing cytoprotection fromthe cytotoxic and genetic effects of ionizing radiation and forproviding cytoprotection from cytotoxic effects of chemotherapeuticagents, the specific dose of compound according to the invention toobtain therapeutic benefit will, of course, be determined by theparticular circumstances of the individual patient including, the size,weight, age and sex of the patient. Also determinative will be thenature and stage of the disease (or cell damage) and the diseaseaggressiveness (or dose of ionizing radiation or chemotherapeuticagent), and the route of administration. For example, a daily dosage offrom about 0.05 to about 50 mg/kg/day may be utilized. Higher or lowerdoses are also contemplated.

For radioprotective administration, the compounds of the inventionshould be administered far enough in advance of the therapeuticradiation such that the compound is able to reach the normal cells ofthe subject in sufficient concentration to exert a radioprotectiveeffect on the normal cells. The pharmacokinetics of specific compoundsmay be determined by means known in the art and tissue levels of acompound in a particular individual may be determined by conventionalanalyses.

The compound may be administered as much as about 24 hours, preferablyno more than about 18 hours, prior to administration of the radiation.In one embodiment, the therapy is administered at least about 6-12 hoursbefore administration of the therapeutic radiation. Most preferably, thecompound is administered once at about 18 hours and again at about 6hours before the radiation exposure. One or more aryl or heteroarylpropene amides may be administered simultaneously, or different aryl orheteroaryl propene amides may be administered at different times duringthe treatment.

Where the therapeutic radiation is administered in serial fashion, it ispreferable to intercalate the administration of one or moreradioprotective compounds within the schedule of radiation treatments.As above, different radioprotective compounds of the invention may beadministered either simultaneously or at different times during thetreatment. Preferably, an about 24-hour period separates administrationof the radioprotective compound and the therapeutic radiation. Morepreferably, the administration of the radioprotective compound and thetherapeutic radiation is separated by about 6 to 18 hours. This strategywill yield significant reduction of radiation-induced side effectswithout affecting the anticancer activity of the therapeutic radiation.

For example, therapeutic radiation at a dose of 0.1 Gy may be givendaily for five consecutive days, with a two-day rest, for a total periodof 6-8 weeks. One or more aryl or heteroaryl propene amides may beadministered to the subject 18 hours previous to each round ofradiation. It should be pointed out, however, that more aggressivetreatment schedules, i.e., delivery of a higher dosage, is contemplatedaccording to the present invention due to the protection of the normalcells afforded by the radioprotective compounds. Thus, theradioprotective effect of the compound increases the therapeutic indexof the therapeutic radiation, and may permit the physician to safelyincrease the dosage of therapeutic radiation above presently recommendedlevels without risking increased damage to the surrounding normal cellsand tissues.

The radioprotective compounds of the invention are further useful inprotecting normal bone marrow cells from radiologic treatments designedto destroy hematologic neoplastic cells or tumor cells which havemetastasized into the bone marrow. Such cells include, for example,myeloid leukemia cells. The appearance of these cells in the bone marrowand elsewhere in the body is associated with various disease conditions,such as the French-American-British (FAB) subtypes of acute myelogenousleukemias (AML), chronic myeloid leukemia (CML), and acute lymphocyticleukemia (ALL).

CML, in particular, is characterized by abnormal proliferation ofimmature granulocytes (e.g., neutrophils, eosinophils, and basophils) inthe blood, bone marrow, spleen, liver, and other tissues andaccumulation of granulocytic precursors in these tissues. The subjectwho presents with such symptoms will typically have more than 20,000white blood cells per microliter of blood, and the count may exceed400,000. Virtually all CML patients will develop “blast crisis”, theterminal stage of the disease during which immature blast cells rapidlyproliferate, leading to death.

Other subjects suffer from metastatic tumors, and require treatment withtotal body irradiation (TBI). Because TBI will also kill the subject'shematopoietic cells, a portion of the subject's bone marrow is removedprior to irradiation for subsequent reimplantation. However, metastatictumor cells are likely present in the bone marrow, and reimplantationoften results in a relapse of the cancer within a short time.

Subjects presenting with neoplastic diseases of the bone marrow ormetastatic tumors may be treated by removing a portion of the bonemarrow (also called “harvesting”), purging the harvested bone marrow ofmalignant stem cells, and reimplanting the purged bone marrow.Preferably, the subject is treated with radiation or some otheranti-cancer therapy before the autologous purged bone marrow isreimplanted.

Thus, the invention provides a method of reducing the number ofmalignant cells in bone marrow, comprising the steps of removing aportion of the subject's bone marrow, administering an effective amountof at least one radioprotective compound according to the presentinvention and irradiating the treated bone marrow with a sufficient doseof ionizing radiation such that malignant cells in the bone marrow arekilled. As used herein, “malignant cell” means any uncontrollablyproliferating cell, such a tumor cell or neoplastic cell. Theradioprotective compounds protect the normal hematopoietic cells presentin the bone marrow from the deleterious effects of the ionizingradiation. The compounds also exhibit a direct killing effect on themalignant cells. The number of malignant cells in the bone marrow issignificantly reduced prior to reimplantation, thus minimizing theoccurrence of a relapse.

Preferably, each aryl or heteroaryl propene amide is administered in aconcentration from about 0.25 to about 100 micromolar; more preferably,from about 1.0 to about 50 micromolar; in particular from about 2.0 toabout 25 micromolar. Particularly preferred concentrations are 0.5, 1.0and 2.5 micromolar and 5, 10 and 20 micromolar. Higher or lowerconcentrations may also be used.

The radioprotective compounds may be added directly to the harvestedbone marrow, but are preferably dissolved in an organic solvent such asdimethylsulfoxide (DMSO). Pharmaceutical formulations of aryl andheteroaryl propene amides such as are described in more detail below mayalso be used.

Preferably, the radioprotective compound is added to the harvested bonemarrow about 20 hours prior to radiation exposure, preferably no morethan about 24 hours prior to radiation exposure. In one embodiment, theradioprotective compound is administered to the harvested bone marrow atleast about 6 hours before radiation exposure. One or more compounds maybe administered simultaneously, or different compounds may beadministered at different times. Other dosage regimens are alsocontemplated.

If the subject is to be treated with ionizing radiation prior toreimplantation of the purged bone marrow, the subject may be treatedwith one or more radioprotective compounds prior to receiving theionizing radiation dose, as described above.

A subject may also be exposed to ionizing radiation from occupation orenvironmental sources, as discussed in the background section. Forpurposes of the invention, the source of the radiation is not asimportant as the type (i.e., acute or chronic) and dose level absorbedby the subject. It is understood that the following discussionencompasses ionizing radiation exposures from both occupational andenvironmental sources.

Subjects suffering from effects of acute or chronic exposure to ionizingradiation that are not immediately fatal are said to have remediableradiation damage. Such remediable radiation damage can reduced oreliminated by the compounds and methods of the present invention.

An acute dose of ionizing radiation which may cause remediable radiationdamage includes a localized or whole body dose, for example, betweenabout 10,000 millirem (0.1 Gy) and about 1,000,000 millirem (10 Gy) in24 hours or less, preferably between about 25,000 millirem (0.25 Gy) andabout 200,000 (2 Gy) in 24 hours or less, and more preferably betweenabout 100,000 millirem (1 Gy) and about 150,000 millirem (1.5 Gy) in 24hours or less.

A chronic dose of ionizing radiation which may cause remediableradiation damage includes a whole body dose of about 100 millirem (0.001Gy) to about 10,000 millirem (0.1 Gy), preferably a dose between about1000 millirem (0.01 Gy) and about 5000 millirem (0.05 Gy) over a periodgreater than 24 hours, or a localized dose of 15,000 millirem (0.15 Gy)to 50,000 millirem (0.5 Gy) over a period greater than 24 hours.

The invention therefore provides a method for treating individuals whohave incurred remediable radiation damage from acute or chronic exposureto ionizing radiation, comprising reducing or eliminating the cytotoxiceffects of radiation exposure on normal cells and tissues byadministering an effective amount of at least one radioprotectivecompound. The compound is preferably administered in as short a time aspossible following radiation exposure, for example between 0-6 hoursfollowing exposure.

Remediable radiation damage may take the form of cytotoxic and genotoxic(i.e., adverse genetic) effects in the subject. In another embodiment,there is therefore provided a method of reducing or eliminating thecytotoxic and genotoxic effects of radiation exposure on normal cellsand tissues, comprising administering an effective amount of at leastone radioprotective compound prior to acute or chronic radiationexposure. The compound may be administered, for example about 24 hoursprior to radiation exposure, preferably no more than about 18 hoursprior to radiation exposure. In one embodiment, the compound isadministered at least about 6 hours before radiation exposure. Mostpreferably, the compound is administered at about 18 and again at about6 hours before the radiation exposure. One or more radioprotectivecompounds may be administered simultaneously, or differentradioprotective compounds may be administered at different times.

When multiple acute exposures are anticipated, the radioprotectivecompounds of the invention may be administered multiple times. Forexample, if fire or rescue personnel must enter contaminated areasmultiple times, radioprotective compounds of the invention may beadministered prior to each exposure. Preferably, an about 24-hour periodseparates administration of the compound and the radiation exposure.More preferably, the administration of radioprotective compounds and theradiation exposure is separated by about 6 to 18 hours. It is alsocontemplated that a worker in a nuclear power plant may be administeredan effective amount of a radioprotective compound of the invention priorto beginning each shift, to reduce or eliminate the effects of exposureto ionizing radiation.

If a subject is anticipating chronic exposure to ionizing radiation, theradioprotective compound may be administered periodically throughout theduration of anticipated exposure. For example, a nuclear power plantworker or a soldier operating in a forward area contaminated withradioactive fallout may be given the radioprotective compound every 24hours, preferably every 6-18 hours, in order to mitigate the effects ofradiation damage. Likewise, the radioprotective compound may beperiodically administered to civilians living in areas contaminated byradioactive fallout until the area is decontaminated or the civiliansare removed to a safer environment.

For providing cytoprotection from cytotoxic effects of chemotherapeuticagents, the schedule of administration of the cytotoxic drug, ie.,mitotic phase cell cycle inhibitor or topoisomerase inhibitor, can beany schedule with the stipulation that the aryl or heteroaryl propeneamide is administered prior to the cytotoxic drug. The cytoprotectivecompound should be administered far enough in advance of the cytotoxicdrug such that the former is able to reach the normal cells of thepatient in sufficient concentration to exert a cytoprotective effect onthe normal cells. Again, individual drug pharmacokinetics and bloodlevels of a specific drug in a specific patient are factors that may bedetermined by methods known in the art.

In one embodiment, the cytoprotective compound is administered at leastabout 4 hours before administration of the cytotoxic drug. The compoundmay be administered as much as about 48 hours, preferably no more thanabout 36 hours, prior to administration of the cytotoxic drug. Mostpreferably, the compound is administered about 24 hours before thecytotoxic drug. The compound may be administered more or less than 24hours before the cytotoxic effect, but the protective effect of thecompounds is greatest when administered about 24 hours before thecytotoxic drug. One or more cytotoxic drugs may be administered.Similarly, one or more of the aryl or heteroaryl propene amides may becombined.

Where the cytotoxic drug or drugs is administered in serial fashion, itmay prove practical to intercalate cytoprotective compounds of theinvention within the schedule with the caveat that a 4-48 hour period,preferably a 12-36 hour period, most preferably a 24 hour period,separates administration of the two drug types. This strategy will yieldpartial to complete eradication of cytotoxic drug side effects withoutaffecting anticancer activity.

For example, the mitotic inhibitor may be given daily, or every fourthday, or every twenty-first day. The aryl or heteroaryl propene amide maybe given 24 hours previous to each round of inhibitor administration,both as a cytoprotective agent and as an antitumor agent.

The compounds of the invention may be administered for therapeuticeffect by any route, for example enteral (e.g., oral, rectal,intranasal, etc.) and parenteral administration. Parenteraladministration includes, for example, intravenous, intramuscular,intraarterial, intraperitoneal, intravaginal, intravesical (e.g., intothe bladder), intradermal, topical, subcutaneous or sublingualadministration. Also contemplated within the scope of the invention isthe instillation of drug in the body of the patient in a controlledformulation, with systemic or local release of the drug to occur at alater time. For anticancer use, the drug may be localized in a depot forcontrolled release to the circulation, or local site of tumor growth.

The compounds of the invention may be administered in the form of apharmaceutical composition, in combination with a pharmaceuticallyacceptable carrier. The active ingredient in such formulations maycomprise from 0.1 to 99.99 weight percent. By “pharmaceuticallyacceptable carrier” is meant any carrier, diluent or excipient which iscompatible with the other ingredients of the formulation and notdeleterious to the recipient.

The active agent is preferably administered with a pharmaceuticallyacceptable carrier selected on the basis of the selected route ofadministration and standard pharmaceutical practice. The active agentmay be formulated into dosage forms according to standard practices inthe field of pharmaceutical preparations. See Alphonso Gennaro, ed.,Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack PublishingCo., Easton, Pa. Suitable dosage forms may comprise, for example,tablets, capsules, solutions, parenteral solutions, troches,suppositories, or suspensions.

For parenteral administration, the active agent may be mixed with asuitable carrier or diluent such as water, an oil (particularly avegetable oil), ethanol, saline solution, aqueous dextrose (glucose) andrelated sugar solutions, glycerol, or a glycol such as propylene glycolor polyethylene glycol. Solutions for parenteral administrationpreferably contain a water-soluble salt of the active agent. Stabilizingagents, antioxidizing agents and preservatives may also be added.Suitable antioxidizing agents include sulfite, ascorbic acid, citricacid and its salts, and sodium EDTA. Suitable preservatives includebenzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. Thecomposition for parenteral administration may take the form of anaqueous or nonaqueous solution, dispersion, suspension or emulsion.

For oral administration, the active agent may be combined with one ormore solid inactive ingredients for the preparation of tablets,capsules, pills, powders, granules or other suitable oral dosage forms.For example, the active agent may be combined with at least oneexcipient such as fillers, binders, humectants, disintegrating agents,solution retarders, absorption accelerators, wetting agents absorbentsor lubricating agents. According to one tablet embodiment, the activeagent may be combined with carboxymethylcellulose calcium, magnesiumstearate, mannitol and starch, and then formed into tablets byconventional tableting methods.

The practice of the invention is illustrated by the followingnon-limiting examples. Representative compounds are listed in Table 5.

EXAMPLE 1(E)-N-(4-Methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propen-amide

A solution of 4-methoxyphenylamino-3-oxoprpanoic acid and2,4,6-trimethoxy benzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 198-200° C, was therebyobtained in 78% yield.

NMR (DMSO-d6),δ3.78 (s, 3H), 3.86 (s, 6H), 3.88 (s, 3H), 6.13 (s, 2H),6.85 (d, 1H vinylic), 6.88-7.56 (m, aromatic), 8.12 (d, 1H vinylic,J=16.0 Hz).

EXAMPLE 2 (E)-N-(4-Methophenyl)-3-(2,6-dimethoxypheyl)-2-propenamide

A solution of 4-methoxyphenylamino-3-oxoprpanoic acid and 2,6-dimethoxybenzaldehyde was reacted according to General Procedure 1. The titlecompound, melting point 203-204° C., was thereby obtained in 63% yield.

NMR (DMSO-d6), δ 3.80 (s, 3H), 3.90 (s, 6H), 7.0 (d, 1H vinylic, J=15.5Hz), 6.57-7.57 (m, aromatic), 8.17 (d, 1H vinylic, J=15.5 Hz).

EXAMPLE 3(E)-N-(4-Methoxy-3-nitropheneyl-3-(3,4,5-trimethoxphenyl-2-propenamide

A solution of 4-methoxy-3-nitroaniline and 3,4,5-trimethoxy cinnamicacid was reacted according to General Procedure 3. The title compound,melting point 186-189° C., was thereby obtained in 35% yield.

NMR (DMSO-d6), δ 3.84 (s, 3H), 3.90 (s, 9H), 7.2 (d, 1H vinylic, J=16.0Hz), 7.37-8.20 (m, aromatic).

EXAMPLE 4(E)-N-(4-Methoxy-3-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide

A solution of N-(4-methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide (Example 3) (1.3 mmol) in acetone/water (10:5) was heatedto 50° C. After 30 minutes, sodium hydrosulfite (Na₂S₂O₄) (26.3 mmol)was added slowly. The resulting mixture was heated at reflux for onehour and then cooled to room temperature. Water was added to the cooledreaction mixture. The product was isolated by extraction with ethylacetate. The organic layer washed with 10% aqueous NaHCO₃ and then driedover anhydrous Na₂SO₄. The solvent was removed under reduced pressure.The crude product thereby obtained was recrystallized from 2-propanol toyield the title compound, melting point 202-204° C., in 32% yield.

NMR (DMSO-d6) δ 3.82 (s, 3H), 3.88 (s, 6H), 6.45 (d, 1H vinylicJ=15.5Hz), 6.70-7.45 (m, aromatic), 7.60 (d, 1H vinylicJ=15.5 Hz).

EXAMPLE 5(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,4,6-trimethoxyphenyl-2-propenamide

A solution of 4-methoxy-3-nitrophenylamino-3-oxopropanoic acid and2,4,6-trimethoxy benzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 138-140° C., was therebyobtained in 58% yield.

NMR (DMSO-d6), δ 3.84 (s, 3H), 3.86 (s, 6H), 3.92 (s,3H), 6.18 (s, 2H),7.12 (d, 1H vinylic), 6.94-8.17 (m, aromatic).

EXAMPLE 6(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide

A solution of N-(4-methoxy-3-nitrophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide (example 5) (1.3mmol) in acetone/water (10:5) was heatedto 50° C. After 30 min, sodium hydrosulfite (Na₂S₂O₄) 26.3 mmol) wasadded slowly. The resulting mixture was heated at reflux (50° C.) forone hour. The mixture was then cooled to room temperature and water wasadded. The product was isolated by extraction with ethyl acetate. Theorganic layer washed with 10% aqueous NaHCO₃ and then dried overanhydrous Na₂SO₄. The solvent was removed under reduced pressure. Thecrude product thereby obtained was recrystallized from 2-propanol toyield the title compound, melting point 92-94° C., in 48% yield.

NMR (DMSO-d6) δ 3.82 (s, 3H), 3.88 (s, 6H), 6.13 (s, 2H), 6.45 (d, 1Hvinylic, J=15.5 Hz), 6.70-7.45 (m, aromatic), 7.60 (d, 1H vinylic J=15.5Hz).

EXAMPLE 7(E)-N-(4-Methoxy-3-nitrophenyl)-3-(2,3,4,5,6-pentafluorophenyl)-2-propenamide

A solution of 4-methoxy-3-nitrophenylamino-3-oxopropanoic acidand2,3,4,5,6-pentafluorobenzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 265-266° C., was therebyobtained in 48% yield.

NMR (DMSO-d6), δ 3.80 (s, 3H), 7.25 (d, 1H vinylic), 7.60 (d, 1Hvinylic), 7.20-7.73 (m, aromatic).

EXAMPLE 8(E)-N-(4-Methoxy-3-nitrophenyl)-3-(3-fluoro-4-nitrophenyl)-2-propenamide

A solution of 4-methoxy-3-nitrophenylamino-3-oxopropanoic acid and3-fluoro-4-nitrobenzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 263-265° C., was therebyobtained in 57% yield.

NMR (DMSO-d6), δ 3.78 (s, 3H), 3.80 (s, 6H), 7.10 (d, 1H vinylic), 7.62(d, 1H vinylic), 7.20-7.85 (m, aromatic).

EXAMPLE 9(E)-N-(4-Methoxy-3-aminophenyl)-3-(3-fluoro-4-aminophenyl)-2-propenamide

A solution ofN-(4-methoxy-3-nitrophenyl)-3-(3-fluoro-4-nitropheny)-2-propenamide(Example 8) (1.3 mmol) in acetone-water (10:5) was reacted according tothe procedure as described in the Example 6. The title compound, meltingpoint 170-172° C., was thereby obtained in 47% yield.

NMR (DMSO-d6) δ 3.84 (s, 3H), 5.10 (s, 2H), 5.35 (s, 2H), 6.90 (d, 1Hvinylic),7.60 (d, 1H vinylic), 6.70-7.45 (m, aromatic).

EXAMPLE 10(E)-N-(4-Methoxy-3-trifluoroacetamidophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide

A solution of trifluoroacetic anhydride (20 mmol) and(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide(10 mmol) ( Example 6) in dry DCM (25 mL) was stirred for 2 hours atambient temperature. Then, the reaction mixture was concentrated undervacuum and the crude product obtained was purified by washing withdiethyl ether to yield the desired product in 43% yield.

EXAMPLE 11(E)-N-(3-Hydoxy-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide

A solution of 3-hydroxy-4-methoxyphenylamino-3-oxopropanoic acid and2,4,6-trimethoxybenzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 188-189° C., was therebyobtained in 52% yield.

NMR (DMSO-d6), δ 3.82 (s, 3H), 3.84 (s, 6H), 3.94 (s, 3H), 6.12 (s 2H),7.18 (d, 1H vinylic), 7.64 (d,1H vinylic), 7.10-7.87 (m, aromatic).

EXAMPLE 12(E)-N-(4-Bromophenyl)-3-(3-methoxy-4fluorophenyl)-2-propenamide

A solution of 4-bromophenylamino-3-oxopropanoic acid and3-methoxy-4-fluorobenzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 163-165° C., was therebyobtained in 52% yield.

NMR (DMSO-d6), δ 3.80 (s, 3H), 7.0 (d, 1H vinylic), 7.68 (d 1H vinylic),7.25-7.88 (m, aromatic).

EXAMPLE 13(E)-N-(4-Bromophenyl)-3-(3-cyano-4-fluorophenyl)-2-propenamide

A solution of 4-bromophenylamino-3-oxopropanoic acid and3-cyano-4-fluorobenzaldehyde was reacted according to GeneralProcedure 1. The title compound, melting point 205-210° C., was therebyobtained in 57% yield.

NMR (DMSO-d6), 5 7.15 (d, 1H vinylic), 7.72 (d 1H vinylic), 7.25-8.10(m, aromatic).

EXAMPLE 14(E)-N-(4-Bromophenyl)-3-(3-carboxy-4-fluorophenyl)-2-propenamide

(E)-N-(4-Bromophenyl)-3-(3-cyano-4-fluorophenyl)-2-propenamide(Example13) (1 gm) was dissolved in acetic acid (10 mL). Aqueous sulfuric acid(50%, 10 mL) was then slowly added to the acetic acid solution. Theresulting solution was refluxed for 3 hours, then cooled and poured intocold water. The resulting solid precipitate was collected by filtrationand purified by column chromatography over silica to yield 32% of thedesired product.

EXAMPLE 15 Effect of Aryl and Heteroaryl Propene Amides on Tumor CellLines

The effect of the aromatic acrylamides on normal fibroblasts and ontumor cells was determined by the assay described by Latham etal.,Oncogene 12:827-837 (1996). Normal diploid lung human fibroblasts(HFL-1) or tumor cells (BT20(breast cancer), DLD1 (colorectal cancer),DU145 (prostate cancer and K562 (chronic myelogenous leukemia )) wereplated in 6-well dishes at a cell density of 1.0 ×10 ⁵cells per 35-mm²well. The plated cells were treated 24 hours later with a compound ofthe invention, dissolved in DMSO at multiple concentrations ranging from100 nM to 10 μM. The total number of viable cells was determined 96hours later by trypsinizing the wells and counting the number of viablecells, as determined by trypan blue exclusion, using a hemacytometer.Normal HFL cells were treated with the same compounds under the sameconditions of concentration and time. The normal cells displayed growthinhibition but no appreciable cell death.

Representative examples of activities of compounds of the invention incell lines: BT20(breast cancer), DLD1 (colorectal cancer), DU145(prostate cancer and K562 (chronic myelogenous leukemia ) are listed inTable 5.

TABLE 5 Example # Structure BT20 DU145 K562 DLD1 1

+++ +++ +++ +++ 2

+ + + + 3

NT NT NT NT 4

+ + + + 5

NT NT NT NT 6

+++ +++ +++ +++ 7

++ ++ ++ ++ 8

NT NT NT NT 9

++ ++ ++ ++ 10

+++ +++ +++ +++ 11

++++ ++++ ++++ ++++ 12

++ ++ ++ ++ 13

++ ++ ++ ++ 14

NT NT NT NT + denotes activity at >10 μM; ++ denotes activity at 10 μM;+++ denotes activity at 100 nM; ++++ denotes activity at <100 nm; NT =not tested.

EXAMPLE 16 Induction of Apoptosis in Tumor Cells

The following assay demonstrates the apoptotic activity of the compoundsof the invention against tumor cells.

The caspases and the ICE-family proteases are cysteine proteases whichare activated during apoptosis (Patel et al., FASEB 10:587-597, 1996).The cleavage of poly(ADP-ribose) polymerase (PARP), which is a target ofcaspase-3, apopain, and several other activated proteases, is a widelyused and accepted marker for apoptosis (Nicholson et al., Nature376(6533):37-43, 1995; Lippke et al., J. Biol. Chemistry 271:1825,1996).

For this assay, BT20 cells, an estrogen receptor negative breastcarcinoma, and HFL-1 cells, normal lung fibroblasts, are treated with acompound according to the present invention at a final concentration of20 μM in DMSO for 96 hours. The cells are then lysed in RIPA buffer and100 μg of total cellular protein from each sample is resolved on a 10%SDS-polyacrylamide gel. The proteins are then Western blotted ontoPROTRAN filter paper (S/S) and the filter is then probed with antibody(Boehringer Mannheim) specific for PARP. This antibody recognizes boththe 116 kDa full length PARP and the 83 kDa cleaved product. The assaywill show whether the test compound specifically activates caspases inthe treated breast carcinoma cell line and not in the normal cell line.The western blot will show whether only the test compound-treated BT20cells displayed the presence of the 83 kDa PARP cleavage product. TheHFL-1 cells treated in a similar manner as controls, will show nocleavage of the full length PARP. BT20 cells treated with DMSO as acontrol for the same amount of time will also show no activation of theapoptotic pathway. These results will show that the compounds of theinvention selectively kill cancer cells by activating the apoptoticpathway as indicated by the activation of the cysteine proteases, amolecular marker for apoptosis. Cells which are not tumorigenic will notundergo apoptosis but may become growth arrested at concentrationssignificantly higher than the concentration necessary for tumor celldeath.

EXAMPLE 17 Radioprotective Effects of Aryl and Heteroaryl Propene Amideson Cultured Normal Cells

The radioprotective effects of compounds of the invention are evaluatedon cultured normal cells as follows:

HFL-1 cells, which are normal diploid lung fibroblasts, are plated into24 well dishes at a cell density of 3000 cells per 10 mm² in DMEMcompleted with 10% fetal bovine serum and antibiotics. The testcompounds are added to the cells 24 hours later in select concentrationsfrom 2.5 μM and 10.0 μM, inclusive, using DMSO as a solvent. Controlcells are treated with DMSO alone. The cells are exposed to the testcompound or DMSO for 24 hours.

The cells are then irradiated with 10 Gy of ionizing radiation (IR)using a J. L. Shepherd Mark I, Model 30-1 Irradiator equipped with¹³⁷cesium as a source. After irradiation, the medium on the test andcontrol cells is removed and replaced with fresh growth medium withoutthe test compounds or DMSO. The irradiated cells are incubated for 96hours and then duplicate wells are trypsinized and replated onto 100 mm²tissue culture dishes. The replated cells are grown under normalconditions with one change of fresh medium for 2 weeks. The number ofcolonies from each 100 mm² culture dish, which represents the number ofsurviving cells, may be determined by staining the dishes as describedbelow.

In order to visualize and count the colonies derived from the clonaloutgrowth of individual protected cells, the medium is removed and theplates are washed one time with room temperature phosphate bufferedsaline. The cells are stained with a 1:10 diluted Modified Geimsastaining solution (Sigma) for 20 minutes. The stain is removed, and theplates are washed with tap water. The plates are air-dried, the numberof colonies from each plate is counted and the average from duplicateplates is determined. Radioprotective activity of x-fold protection isdetermined by dividing the average number of colonies from the testplates by the average number of colonies counted on the control plates.

EXAMPLE 18 Protection of Mice from Radiation Toxicity by Pre-Treatmentwith Aryl and Heteroaryl Propene Amides

C57 black mice age 10-12 weeks (Taconic) are divided into treatmentgroups of 10 mice each and given intraperitoneal injections of 200micrograms of an aryl or heteroaryl propene amide dissolved in DMSO (a10 mg/Kg dose, based on 20 g mice). The injections are given 18 and 6hours before irradiation with 8 Gy gamma radiation. A control group of10 animals receives 8 Gy gamma radiation alone. Mortality of control andexperimental groups is assessed for 40 days after irradiation.

EXAMPLE 19 Radioprotective Effect of Aryl and Heteroaryl Propene Amidesin Mice when Given After Radiation Exposure

C57 B6/J mice age 10-12 weeks (Taconic) are divided into treatmentgroups and one control group of 10 mice each. Each treatment groupreceives intraperitoneal injections of 200 micrograms of aradioprotective compound of the invention dissolved in DMSO (a 10 mg/Kgdose, based on 20 g mice) 15 minutes after irradiation with 8 Gy gammaradiation. The control group receives 8 Gy gamma radiation alone.Mortality of control and treatment groups are assessed for 40 days afterirradiation.

EXAMPLE 20 Effect of Exposure to Ionizing Radiation on Normal andMalignant Hematopoietic Progenitor Cell Growth After Pretreatment withAryl and Heteroaryl Propene Amides

The effect of ionizing radiation on normal and malignant hematopqieticprogenitor cells which are pretreated with aryl and heteroaryl propeneamides is investigated by assessing cloning efficiency and developmentof the pretreated cells after irradiation.

To obtain hematopoietic progenitor cells, human bone marrow cells (BMC)or peripheral blood cells (PB) are obtained from normal healthy, oracute or chronic myelogenous leukemia (AML, CML), volunteers byFicoll-Hypaque density gradient centrifugation, and are partiallyenriched for hematopoietic progenitor cells by positively selectingCD34⁺ cells with immunomagnetic beads (Dynal A. S., Oslo, Norway). TheCD34⁺ cells are suspended in supplemented alpha medium and incubatedwith mouse anti-HPCA-I antibody in 1:20 dilution, 45 minutes, at 4° C.with gentle inverting of tubes. Cells are washed 3× in supplementedalpha medium, and then incubated with beads coated with the Fc fragmentof goat anti-mouse IgG1 (75 μL of immunobeads/10⁷ CD34⁺cells). After 45minutes of incubation (4° C.), cells adherent to the beads arepositively selected using a magnetic particle concentrator as directedby the manufacturer.

2×10⁴ CD34⁺ cells are incubated in 5 mL polypropylene tubes (FisherScientific, Pittsburgh, Pa.) in a total volume of 0.4 mL of Iscove'smodified Dulbecco's medium (IMDM) containing 2% human AB serum and 10 mMHepes buffer. A compound of the invention, for example(E)-N-(4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide or(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-triiethoxyphenyl)-2-propenamide,at three different concentrations (2.5 μM, 5.0 μM and 10.0 μM) in DMSOis added separately to the cells. Control cells receive DMSO alone. Thecells are incubated for 20-24 hours and irradiated with 5 Gy or 10 Gy ofionizing radiation. Immediately after irradiation, the medium is removedand replaced with fresh medium without the test compound or DMSO.Twenty-four hours after irradiation, the treatment and control cells areprepared for plating in plasma clot or methylcellulose cultures. Cells(1×10⁴ CD34⁺ cells per dish) are not washed before plating.

Assessment of the cloning efficiency and development of the treatedhematopoietic progenitor cells are carried out essentially as reportedin Gewirtz et al., Science 242, 1303-1306 (1988), the entire disclosureof which is incorporated herein by reference.

EXAMPLE 21 Bone Marrow Purging with Ionizing Radiation AfterPretreatment with an Aryl or Heteroaryl Propene Amide

Bone marrow is harvested from the iliac bones of a subject under generalanesthesia in an operating room using standard techniques. Multipleaspirations are taken into heparinized syringes. Sufficient marrow iswithdrawn so that the subject will be able to receive about 4×10⁸ toabout 8×10⁸ processed marrow cells per kg of body weight. Thus, about750 to 1000 mL of marrow is withdrawn. The aspirated marrow istransferred immediately into a transport medium (TC-199, Gibco, GrandIsland, N.Y.) containing 10,000 units of preservative-free heparin per100 mL of medium. The aspirated marrow is filtered through threeprogressively finer meshes to obtain a cell suspension devoid ofcellular aggregates, debris and bone particles. The filtered marrow isthen processed further into an automated cell separator (e.g., Cobe 2991Cell Processor) which prepares a “buffy coat” product, (i.e., leukocytesdevoid of red cells and platelets). The buffy coat preparation is thenplaced in a transfer pack for further processing and storage. It may bestored until purging in liquid nitrogen using standard procedures.Alternatively, purging can be carried out immediately, then the purgedmarrow may be stored frozen in liquid nitrogen until it is ready fortransplantation.

The purging procedure is carried out as follows. Cells in the buffy coatpreparation are adjusted to a cell concentration of about 2×10⁷/mL inTC-199 containing about 20% autologous plasma. A compound of theinvention; for example 2.5 to 10 micromolar of either(E)-N-(4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propen-amide or(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamidein DMSO is added to the transfer packs containing the cell suspensionand incubated in a 37° C. water bath for 20-24 hours with gentleshaking. The transfer packs are then exposed to 5-10 Gy ionizingradiation. Recombinant human hematopoietic growth factors, e.g., rH IL-3or rH GM-CSF, may be added to the suspension to stimulate growth ofhematopoietic neoplasms and thereby increase their sensitivity toionizing radiation.

The cells may then either be frozen in liquid nitrogen or washed once at4° C. in TC-199 containing about 20% autqlogous plasma. Washed cells arethen infused into the subject. Care must be taken to work under sterileconditions wherever possible and to maintain scrupulous aseptictechniques at all times.

EXAMPLE 22 Protection of Normal Human Fibroblasts from PaclitaxelCytotoxicity by Aryl and Heteroaryl Propene Amides

HFL-1 cells are plated at a cell density of 1.0×10⁵ per well 24 hoursprior to drug addition. Cells are pretreated with a cytoprotectivecompound of formula I (2.0 μM) for 8 hours and then exposed topaclitaxel (250 μM). Other cells are treated with paclitaxel alone, orboth agents simultaneously. Cells are enumerated by Trypan blueexclusion using a hematocytometer 96 hours after exposure to paclitaxel.Cytoprotective activity may be compared by comparing the number ofviable cells following treatment with a cytoprotective compound of theinvention and paclitaxel, divided by the number of viable cellsremaining after treatment with paclitaxel alone.

EXAMPLE 23 Protection of Normal Human Fibroblasts from Anticancer AgentCytotoxicity

HFL-1 cells are plated at a cell density of 1.0×10⁵ in 1 mL of medium.Twenty-four hours following plating, 2.0 μM of a cytoprotective compoundof the invention was added to the medium. Following a 24-hourpreincubation with the cytoprotective compound, the various cytotoxicagents (selected from the list below) are added to the cells.

The number of viable cells is determined by Trypan blue exclusion usinga hematocytometer 96 hours after exposure to cytotoxic agent. The“Protection Ratio” is the number of viable cells following treatmentwith a cytoprotective compound of the invention and cytotoxic agent,divided by the number of viable cells remaining after treatment withcytotoxic agent alone. A protection ratio of 2 or more is consideredhighly significant, while a protection ratio of 1.5-2 is considered lesssignificant.

Therapeutic concentration Drug (μM) Mechanism of Action paclitaxel 0.25antimitotic vincristine 0.25 antimitotic camptothecin 0.5 topoisomeraseI inhibitor etoposide 3.0 topoisomerase II inhibitor mitoxantrone 0.3topoisomerase II inhibitor doxorubicin 0.4 topoisomerase II inhibitor5-fluorouracil 20 DNA antimetabolite cisplatin 5.0 alkylating agent

EXAMPLE 24 Protection of Normal Human Fibroblasts from VincristineCytotoxicity by Aryl Propene Amides

HFL-1 cells are treated with 0-250 μM vincristine and, optionally, a 2.0μM preparation of a compound of the invention, either 24 hours before orafter vincristine treatment, or simultaneously with vincristinetreatment. Cell viability is assessed 96 hours after the addition ofvincristine.

EXAMPLE 25 Protection of Mice From Paclitaxel Toxicity Using Aryl andHeteroaryl Propene Amides

ICR female mice age 10-12 weeks (Taconic) are divided into the followingtreatment groups and receive intraperitoneal injections of 50 mg/Kg acompound of the invention, dissolved in DMSO and/or 150 mg/kg paclitaxel(Taxol, Sigma Chemical Co.) dissolved in DMSO. The compound of formula Iis given 24 hours before paclitaxel, 4 hours before paclitaxel, orsimultaneously with paclitaxel. Control animals receive paclitaxel aloneor a compound of the invention alone. Mortality is assessed 48 and 144hours after paclitaxel injection.

EXAMPLE 26 Antitumor and Cytoprotection Assay of Aryl and HeteroarylPropene Amides

A. Antitumor Assay

The aryl and heteroaryl propene amides may be tested for antitumoractivity as follows:

A panel of the following human carcinoma cell lines is plated at a celldensity of 1.0 ×10⁵ cells per well in six culture plates: prostate tumorcell line DU-145; breast tumor cell line BT20; chronic myelogenousleukemia cell line K562; and colorectal carcinoma cell line DLD-1. Thecompounds are added to the cultures at a final concentration of 2.5 μM,and 96 hours later the total number of viable cells is determined bycounting the number of viable cells, as determined by Trypan blueexclusion, using a hematocytometer. The activity of each compound isdetermined by comparing the viable cell number of treated to untreatedcontrols.

B. Cytoprotection Assay

The cytoprotective activity of the compounds may be determined asfollows:

Normal human HFL-1 cells are plated at a cell density of 1.0×10⁵ cellsper well in culture plates. A cytoprotective compound of the inventionis added 24 hours later at a final concentration of 2.0-10 μM. The timeof addition of the cytoprotective compound is designated as time zero.Paclitaxel (250 nM) is added at either time zero, or 24 hours after timezero. The total number of viable cells is determined, as describedabove, after 96 hours of paclitaxel treatment. A cytoprotective compoundis deemed to be active if the number of viable cells following thecombination treatment is higher than the number of cells after treatmentwith paclitaxel alone.

All references cited herein are incorporated by reference. The presentinvention may be embodied in other specific forms without departing fromthe spirit or essential attributes thereof and, accordingly, referenceshould be made to the appended claims, rather than to the foregoingspecification, as indication the scope of the invention.

1. A compound selected from the group consisting of:(E)-N-(4-methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,3,4,5,6-pentafluorophenyl)-2-propenamide;(E)-N-(4-methoxy-3-nitrophenyl)-3-(3-fluoro-4-nitrophenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(3-fluoro-4-aminophenyl)-2-propenamide;(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(2E)-N-[4-methoxy-3-(2,2,2-trifluoroacetylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;and salts of such compounds.
 2. The compound(E)-N-(3-hydroxy-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide,or a salt thereof.
 3. A compound selected from the group consisting of:2-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)aceticacid;2-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)propanoicacid;4-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)butanoicacid;3-({5-[(2E)-3-(2,4,6-trimethoxyphenyl)prop-2-enoylamino]-2-methoxyphenyl}amino)propanoicacid; and salts of such compounds.
 4. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and at least onecompound according to claim 1, 2, or
 3. 5. A method of treating anindividual for a cancer selected from breast cancer, prostate cancer,lung cancer and colorectal cancer comprising administering to saidindividual an effective amount of at least one compound selected fromthe group consisting of:(E)-N-(4-methoxy-3-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,3,4,5,6-pentafluorophenyl)-2-propenamide;(E)-N-(4-bromophenyl)-3-(3-methoxy-4-fluorophenyl)-2-propenamide;(E)-N-(4-bromophenyl)-3-(3-cyano-4-fluorophenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(3-fluoro-4-aminophenyl)-2-propenamide;(E)-N-(4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxyphenyl)-3-(2,6-dimethoxyphenyl)-2-propenamide;(E)-N-(3-hydroxy-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(2E)-N-[4-methoxy-3-(2,2,2-trifluoroacetylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;and salts thereof.
 6. A method of selectively inducing apoptosis oftumor cells selected from the group of tumors consisting of breast,prostate, lung, and colorectal tumors in an individual afflicted withcancer comprising administering to said individual an effective amountof at least one compound selected from the group consisting of:(E)-N-(4-methoxy-3-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-nitrophenyl)-3-(2,3,4,5,6-pentafluorophenyl)-2-propenamide;(E)-N-(4-bromophenyl)-3-(3-methoxy-4-fluorophenyl)-2-propenamide;(E)-N-(4-bromophenyl)-3-(3-cyano-4-fluorophenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(3-fluoro-4-aminophenyl)-2-propenamide;(E)-N-(4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxyphenyl)-3-(2,6-dimethoxyphenyl)-2-propenamide;(E)-N-(3-hydroxy-4-methoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(E)-N-(4-methoxy-3-aminophenyl)-3-(2,4,6-trimethoxyphenyl)-2-propenamide;(2E)-N-[4-methoxy-3-(2,2,2-trifluoroacetylamino)phenyl]-3-(2,4,6-trimethoxyphenyl)prop-2-enamide;and salts thereof.